WO2016091272A1

WO2016091272A1 - System and method for establishing a long-term culture of avian primordial germ cells and uses thereof

Info

Publication number: WO2016091272A1
Application number: PCT/EP2014/003333
Authority: WO
Inventors: Ulrich WERNERY; Chunhai Liu; Jorg KINNE; Renate WERNERY; Ali RIDHA
Original assignee: Central Veterinary Research Laboratory
Priority date: 2014-12-12
Filing date: 2014-12-12
Publication date: 2016-06-16
Also published as: WO2016091385A1

Abstract

The present invention provides a system and method for establishing a long-term culture of avian primordial germ cells and uses thereof. The invention is particularly useful to isolate and cultivate primordial germ cells from an early stage of avian embryos. In particular, the present invention provides a method for establishing a long-term culture of avian primordial germ cells, wherein the primordial germ cells are derived from germinal crescent tissue isolated from an avian embryo at stage 4 to 11, preferably at stage 4 to 10, more preferably at stage 6 to 10, even more preferably at stage 7 to 10. Accordingly, the present invention also provides a respective cell culture and cell line of avian primordial germ cells. The cultured avian primordial germ cells can be used for example to preserve the genetic diversity of avian species or as a targeting vehicle cell for genetic modification for disease resistance or other purposes, and to provide an interspecies or intraspecies germline chimera, e.g. for the purpose of propagation of endangered species in captive breeding programmes. Thus, the avian primordial germ cells can be genetically modified. Furthermore, the present invention also provides a method for maintaining a culture of avian primordial germ cells, as well as a germline chimera and a method for obtaining a germline chimera.

Description

Applicant

Central Veterinary Research Laboratory, Dubai, UAE

SYSTEM AND METHOD FOR ESTABLISHING A LONG-TERM CULTURE OF AVIAN

PRIMORDIAL GERM CELLS AND USES THEREOF

The present invention relates to avian biotechnology and in particular to a system and method for establishing a long-term culture of avian primordial germ cells and uses thereof. The invention is particularly useful to isolate and cultivate primordial germ cells from an early stage of avian embryos. The cultured avian primordial germ cells can be used for example to preserve the genetic diversity of avian species or as a targeting vehicle cell for genetic modification for disease resistance or other purposes, and to provide an interspecies or intraspecies germline chimera, e.g. for the purpose of propagation of endangered species in captive breeding programmes.

Habitat loss, human disturbance and possible hunting have reduced the population of various avian species to extremely small, declining population. For example, the population of the houbara bustard is declining dramatically over the last 20 years due to unsustainable hunting, climate change and habitat degradation, and the houbara bustard is classified as a vulnerable species. Various strategies are applied to propagate the houbara bustard and to preserve their genetic diversity. Captive breeding programmes were established in Morocco, Saudi Arabia, and the UAE, with the aim of releasing captive bred populations to boost the wild population (Propagation of houbara bustard. M. Saint Jaime and Y. Van Heezik. Kegan Paul International, London and New Yolk).

A Houbara breeding project was launched at the Central Veterinary Research Laboratory (CVRL), UAE, in the year 2000, with the aim of the propagation and conservation of houbara bustards via novel biotechnologies, such as avian germ cells manipulation and germline chimera. Germline chimeras were produced by transferring houbara primordial germ cells (PGCs) into domestic poultry species (Wernery U, Liu C, Baskar V, Guerineche Z, Khazanehdari KA, Saleem S, Kinne J, Wernery R, Griffin DK, Chang IK. Primordial germ cell- mediated chimera technology produces viable pure-line houbara bustard offspring: potential for repopulating an endangered species. PLoS One 2010; 5(12):e1 5824). Donor houbara PGCs were freshly isolated from embryos, or short-term cultured in vitro, which are limited by seasonal breeding and egg availability.

Germ cells are the only kind of cells to pass the genetic information to the next generation. They play a crucial role in keeping the continuity of the species. The manipulation of germ cells from different stages of gametogenesis is well documented. Emerging technologies were developed to isolate, characterize, culture, cryopreserve and transplant different kinds of germ cells between individuals of the same kind, or different species (T. Ogawa, I. Dobrinski, M. R. Avarbock, and R. L. Brinster, "Xenogeneic spermatogenesis following transplantation of hamster germ cells to mouse testes," Biology of Reproduction, vol. 60, no. 2, pp. 51 5-521 , 1999; Dobrinski, M. R. Avarbock, and R. L. Brinster, "Transplantation of germ cells from rabbits and dogs into mouse testes," Biology of Reproduction, vol. 61 , no. 5, pp. 1331 -1 339, 1999; T. Shinohara, M. Kato, M. Takehashi et al., "Rats produced by interspecies spermatogonia! transplantation in mice and in vitro microinsemination," Proceedings of the National Academy of Sciences of the United States of America, vol. 1 03, no. 37, pp. 1 3624- 1 3628, 2006; M. Nagano, M. R. Avarbock, E. B. Leonida, C. J. Brinster, and R. L. Brinster, "Culture of mouse spermatogonia! stem cells," Tissue and Cell, vol. 30, no. 4, pp. 389-397, 1 998; T. Ogawa, I. Dobrinski, M. R. Avarbock, and R. L. Brinster, "Transplantation of male germ line stem cells restores fertility in infertile mice," Nature Medicine, vol. 6, no. 1 , pp. 29-34, 2000; Shinohara T, Kato M, Takehashi M, Lee J, Chuma S, Nakatsuji N, Kanatsu- Shinohara M, Hirabayashi M. Rats produced by interspecies spermatogonia! transplantation in mice and in vitro microinsemination. PNAS USA 2006; 103 (37):13624-13628).

Primordial germ cells (PGCs) are the precursors of ova and spermatozoa. Unlike pluripotent embryonic stem (ES) cells, PGCs are considered as unipotent stem cells, and have the potential to differentiate into germ cells lineage and give rise to functioning gametes. Avian primordial germ cells first appear at stage X (Eyal-Giladi H. and Kochav S. From cleavage to primitive streak formation: A complementary normal table and a new look at the first stages of the development of the chick. Developmental Biology, 1 976,49: 321 -327) in the ventral surface of the area pellucida, subsequently translocate from the epiblast to the hypoblast, and then migrate to the extra-embryonic region, referred to as the germinal crescent (Ginsburg M. and Eyal-Giladi H. Temporal and spatial aspects of the gradual migration of primordial germ cells from the epiblast into the germinal crescent in the avian embryo. Journal of Embryo Experimental Morphology, 1986,95: 53-71 ). With the development of the gonadal ridge, the avian PGCs enter into the vascular system, passively migrate to the vicinity of the gonadal ridge, leave the blood circulation, and migrate into the gonadal anlage (Kuwana T. Migration of avian primordial germ cells toward the gonadal anlage. Dev Growth Diff 1 993; 35:237- 243). Unlike mammalian PGCs, which migrate between tissues before colonizing into gonad, on the way of migration from extra-gonad, avian PGCs passively migrate with blood circulation.

This unique migration route is a great advantage for the manipulation of avian PGCs. PGCs mediated-avian germline chimera technology was developed by transferring circulating PGCs, gonadal PGCs, and cryopreserved PGCs within or between a wide range of avian species, for example chicken (Tajima A., Naito M., Yasuda Y. et al. Production of germ line chimera by transfer of primordial germ cells in the domestic chicken {Callus domesticus). Theriogenology, 1 993,40: 509-51 9; Nakamura Y, Usui F, Ono T, Takeda K, Nirasawa K, Kagami H, Tagami T. Germline replacement by transfer of primordial germ cells into partially sterilized embryos in the chicken. Biol Reprod 2010; 83:1 30-137), quail (Chang I. K., Naito M., Kuwana T. et al. Production of germline chimeric quail by transfer of gonadal primordial germ cells preserved in liquid nitrogen. Japanese Poultry Science, 1998, 35:321 -328), duck (Chunhai Liu, Kamal A. Khazanehdari, Vijaya Baskar, Shazia Saleem, Joerg Kinne, Ulrich Wernery and ll-Kuk Chang. Production of Chicken Progeny (Gallus gallus domesticus) from Interspecies Germline Chimeric Duck (Anas domesticus) by Primordial Germ Cell Transfer. Biology of Reproduction, 2012 86 4:101 ), turkey (D'Costa S, Pardue SL, Petitte JN. Production of interspecific embryonic germ line chimeras by the intravascular transfer of gonadal PGCs. Transgenic Res 2002; 1 1 (1 ): 84), and pheasant (Kang SJ, Choi WJ, Park JK, Lee MY, Kim MT, Sohn HS, Lim MJ, Han JY. Development of a pheasant interspecies primordial germ cell transfer to chicken embryo: Effect of donor cell sex on chimeric semen production. Theriogenology 2009; 4:51 9-527). Injection of primordial germ cells (PGCs) into the developing avian embryo, for example injection of previously in vitro genetically engineered PGCs, provides a promising technology, especially because these technologies allow (i) to introduce a predetermined genotype into the germline of a recipient embryo, thereby enabling the animal to pass the desired genotype on to future generations, and (ii) to target transgene integration to specific sites within the genome, which should allow high expression levels of the transgene. However, in order to use this approach, an important prerequisite must be fulfilled: cells must survive to in vitro manipulations, while still maintaining their ability to be incorporated within a recipient embryo, to colonize the germline and then to transmit the modification to the progeny. Therefore, culturing and expanding avian PGCs in vitro is the core of avian germline chimera technology.

Long-term cultures of PGCs thus provide several important advantages, such as sustaining valuable genetic characteristics for example of endangered avian species or of important avian breeding lines that are relied upon in the poultry and egg production industries.

In contrast to long-term cultures of embryonic stem (ES) cells, i.e. pluripotent cells which can proliferate via a self-renew mechanism and which keep their potential to differentiate into all types of cells of their lineage, prolonged cultures of PGCs were only established in the recent years. The pioneer works in in vitro PGC cultures were done with a mouse PGCs culture, and demonstrated some factors which are favorable to the survival and proliferation of mouse PGCs in culture (Resnick JL, Bixler LS, Cheng L, Donovan PJ. Long-term proliferation of mouse primordial germ cells in culture. Nature. 359(6395):550-1 . 1 992), for example cytokines (e.g., FGF₂, LIF, SCF etc.), and feeder cells (e.g., STO, mouse fibroblast, Buffalo Rat liver cells).

However, in the first attempts, PGCs were only cultured on a short-term basis. Once the length in culture extended beyond a short number of days, the cells lost their ability to contribute exclusively to the germline. In these early attempts, PGCs maintained in culture did not proliferate and multiply and in the absence of robust growth, the cultures were "terminal" and could not be maintained indefinitely. Over time, these terminal cell cultures degraded and the cells lost their unique PGC morphology and revert to "embryonic germ cells" (EG cells), which are pluripotent stem cells and have a different morphology compared to PGCs. In 1991 , Matusui et al. firstly cultured mouse post migration PGCs into such pluripotent stem cells, named as embryonic germ cells (EG), on an STO feeder layer, in a medium with the addition of LIF, bFGF, SCF (Matusui Y., Zesebo K. and Brigid L. Derived of pi uri potential Embryonic stem cell from murine primordial germ cells inculture. Cell, 1992, 70:841 -847). Thus, early studies reporting successful cultures of PGCs (mainly post-migration PGCs or gonadal PGCs) usually aimed at obtaining pluripotent EG cells (Shamblott M J, Axelman J, Wang S, Bugg E M, et al, Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc. Natl. Acad. Sci. USA, 1998: 1 3726-1 3731 ; Park T.S. and HanJ.Y. Derivation and characterization of pluripotent embryonic germ cells in chicken. Molecular Reproduction and Development, 2000, 56:475-482).

However, EG cells lost their restriction to the germline, and gained the ability to contribute to somatic tissues when injected into early stages of embryonic development, whereas PGCs are the progenitors of sperm and egg and, thus, PGCs are uniquely attractive to introduce a predetermined genotype specifically into the germline of a recipient embryo. Thus, there is a need for a long-term culture of PGCs wherein the cells maintain their unique PGC properties, i.e. the cells show migration capability in vivo, and are able to give rise to functioning gametes in the recipient testis or ovary.

In 2006, Van de Lavoir cultured chicken circulating PGCs from embryonic blood into a germline competent cell line. These cells could be cultured for prolonged period, showed migration capability in vivo, and gave rise to functioning gametes in the recipient testis or ovary (Van de Lavoir MC, Diamond JH, Leighton PA, Mather-Love QHeyer BS, Bradshaw R, Kerchner A, Hooi LT, Gessaro TM, SwanbergSE, Delany ME and Etches RJ. Germline transmission of genetically modified primordial germ cells. Nature, 441 : 766-769. 2006). This success was confirmed later by many other publications (Macdonald J, Glover JD, Taylor L, SangHM and McGrew MJ. Characterisation and germline transmission of cultured avian primordial germ cells. PLoS One, 5: e1 5518. 201 0; Jin Won Choi, Sujung Kim, Tae Min Kim, Young Min Kim, Hee Won Seo, Tae Sub Park, Jae-Wookjeong, Gwonhwa Song, Jae Yong Han. Basic Fibroblast Growth Factor Activates MEK/ERK Cell Signaling Pathway and Stimulates the Proliferation of Chicken Primordial Germ Cells. PLoS One, 2010).

In the light of the successful chicken PGCs culture, the culture of PGCs of other avian species was also attempted. However, no success was reported so far.

In view of the above, it is the object of the present invention to provide a method and a system for establishing a long-term culture of avian primordial germ cells and uses thereof, which can be applied to a variety of avian species, in particular to endangered avian species, such as the houbara bustard.

This object is achieved by means of the subject-matter set out below and in the appended claims. In light of achievements in chicken PGCs culture, the present inventors invented a culture system for avian PGCs, including a source of starting material. Moreover, the present inventors also found feeder cells and soluble cytokines which are favorable for the survival, proliferation and reprogramming of avian PGCs into a germline competent cell line. The present invention provides a novel system to isolate and cultivate primordial germ cells from the early stage of avian embryos, in particular of endangered wild avian species, e.g. the houbara bustard. Avian PGCs expand in the invented culture system for a prolonged period, keep the migratory capability and enable the generation of germline chimera. These cells can be applied to preserve the genetic diversity of avian species, e.g. the houbara bustard, or as a targeting vehicle cell for genetic modification for disease resistance or for other purposes, and to provide an interspecies or intraspecies germline chimera, e.g. for the purpose of propagation of houbara bustard in captive breeding programmes. Moreover, a culture PGC line from a wild endangered bird is described herein for the first time. The invention thus also provides a new approach for preserving genetic diversity and provides resources for the conservation of endangered birds, e.g. the houbara bustard. Method for establishing a culture of avian primordial germ cells

In a first aspect the present invention provides a method for establishing a long-term culture of avian primordial germ cells, wherein the primordial germ cells are derived from germinal crescent tissue isolated from an avian embryo at stage 4 to 1 1 , preferably at stage 4 to 10, more preferably at stage 6 to 10, even more preferably at stage 7 to 10.

The term "culture" or "cell culture" as used herein refers to cells, which are grown under controlled conditions, generally outside of their natural environment. A "long-term" culture as used herein refers to a culture, which can be extended typically through multiple passages to extend the viability of the culture. In particular, a long-term culture is cultured in vitro typically at least 40 days, at least 60 days, at least 80 days, at least 100 days, at least 120 days, at least 140 days, at least 150 days, at least 1 75 days, at least 200 days, at least 225 days, at least 250 days, at least 275 days, at least 300 days, at least 325 days, at least 350 days, or even longer. In particular, a long-term culture is usually a culture, which is maintained at least until passage 5, or longer.

The "passage" number refers to the number of transfers, i.e. to the number of times that a culture is removed from a culture vessel and placed into another culture vessel. Thereby, the cells undergo a so-called passage or subculture process. By subculturing (passage) the cells are typically kept at a sufficiently low density to stimulate further growth.

The term "primary culture" (or passage 0) refers to the first culture following the isolation of cells from their natural environment, in particular from tissue. Following the first transfer, the cells are referred to as a secondary culture (or passage 1 ). After the second transfer, the cells are referred to as a tertiary culture (or passage 2), and so on. Preferably, a long-term culture according to the present invention is a culture maintained at least until passage 10, more preferably maintained at least until passage 20, even more preferably maintained at least until passage 30, and particularly preferably maintained at least until passage 50.

The term "avian" as used herein refers to any taxonomic rank, e.g. order, family, genus, species, subspecies and race, of an organism of the taxonomic class "aves", such as, but not limited to, bustard, chicken, turkey, duck, goose, ibis, quails, pheasants, parrots, finches, hawks, crows, ostrich, emu and cassowary. The terms "avian", "bird", "aves" or "ava" as used herein are intended to have the same meaning, and will be used indistinctly. In the present invention, preferred avian taxonomic orders are Gruiformes, Ciconii formes, and Fa Icon / ormes, as well as Galliformes and Anseriformes, whereby the taxonomic orders Gruiformes, Ciconiiformes, and Fa/con/formes are more preferred.

Furthermore, preferred birds in the present invention are endangered birds. The term "endangered bird" or "endangered avian species" as used herein refers to a bird, or a species respectively, having at least the status "VU" ("vulnerable", i.e. high risk of endangerment in the wild) in the lUCN Red List of Threatened Species. Preferably, the bird and/or the species has at least the status "EN" ("endangered", i.e. high risk of extinction in the wild), more preferably at least the status "CR" ("critically endangered", i.e. extremely high risk of extinction in the wild), and even more preferably at least the status "EW" ("extinct in the wild", i.e. known only to survive in captivity, or as a naturalized population outside its historic range) in the lUCN Red List of Threatened Species.

The order Gruiformes, which is particularly preferred according to the present invention, includes the family Otididae (Bustards), which is particularly preferred, and the genus Chlamydotis, which is particularly preferred and which comprises two species, houbara bustard {Chlamydotis undulata) and MacQueen's bustard {Chlamydotis macqueenif), whereby in the present invention the houbara bustard {Chlamydotis undulata) is in general particularly preferred. The houbara is a medium sized bustard and includes two subspecies: C. u. undulate found in arid habitats spread across northern Africa, and C. u. fuertaventurae is found on the Canary Islands. In particular the subspecies fuertaventurae of the Canary Islands is highly restricted and endangered. A 1 997 survey found a total population of about 500 birds. MacQueen's bustard is distributed from the east of the Sinai peninsula in Palestine, Arabia, to the Caspian Sea and extending east to the Aral Sea in Mongolia. Birds from the northern populations winter further south in Pakistan (mainly in western Balochistan), and in the dry arid zone of western India. Vagrants have historically been found as far west and north as Britain, and as far south as northern Kerala (R. L. Brinster and J. W. Zimmermann, "Spermatogenesis following male germ-cell transplantation," Proceedings of the National Academy of Sciences of the United States of America, vol. 91 , no. 24, pp. 1 1298-1 1302, 1994). The dividing line between the two Chlamydotis species is the Sinai Peninsula. Based on the rates of divergence of mitochondrial DNA sequences, the two subspecies of the houbara bustard (C. u. undulata and C. u. fuertaventurae) are thought to have separated from a common ancestor around 20 to 25 thousand years ago. The separation from MacQueen's Bustard is older at 430 thousand years ago (Idaghdour, Youssef; Broderick, Damien; Korrida, Amal; Chbel, Faiza (2004). "Mitochondrial control region diversity of the houbara bustard Chlamydotis undulata complex and genetic structure along the Atlantic seaboard of North Africa". Molecular Ecology 13(1 ): 43-54. doi:10.1046/j.1 365-294X.2003.02039.x).

The order Ciconiiformes includes the family Threskiornithidae, which is preferred and which includes the preferred subfamily Threskionithinae and the preferred genus Geronticus which comprises the preferred species Geronticus eremita, also referred to as "northern bald ibis" or "waldrapp", which is a critically endangered bird.

The order Fa Icon i formes includes the preferred family Falconidae, including the preferred subfamily Falconinae, which comprises the preferred genus Falco.

The order Anseriformes (e.g. duck, goose, swan and allies) contains about 1 50 species of birds in three families: the Anhimidae (the screamers), Anseranatidae (the Magpie-goose), and the Anatidae, which includes over 140 species of waterfowl, among them the ducks, geese, and swans. All species in the order are highly adapted for an aquatic existence at the water surface. All are web-footed for efficient swimming (although some have subsequently become mainly terrestrial). The term includes the various strains of ducks, for example Pekin duck and Muscovy duck.

The order Galliformes contains the chicken, turkeys, quails and pheasants. About 256 species are found worldwide. The term includes the various strains of Gallus gallus, or chickens, for example S86N, Valo, White Leghorn, Brown Leghorn, Sussex, New Hampshire, Rhode Island, Ausstralorp, Minorca, Amrox, California Gray, East Lansing, Italian-Partridge-colored, Marans, Barred Rock, Cou Nu Rouge (CNR), GF30, ISA as well as strains of turkeys, pheasants, quails, and other poultry commonly bred. In particular, a "culture of avian primordial germ cells" as used herein refers to a culture wherein the specific properties of the PGCs are maintained, i.e. in the PGCs of the culture the PGC properties can be established as outlined below. This applies in particular for the long- term culture, i.e. even after culturing the cells for a prolonged period of time as described above, the cells are still PGCs as defined in the following.

The term "primordial germ cell" or "PGC" as used herein refers to a cell exhibiting PGC properties, e.g. a morphology typical for PGCs and the cell contributing exclusively to the germline, e.g. in recipient embryos. PGC properties (i.e. PGC phenotype and/or genotype) may be established by:

(1 ) the cell exhibits typical PGC morphological characteristics, e.g. rich granula inside cytoplasm, larger in size (15 - 28 pm);

(2) germline specific genes, in particular VASA (or CVH) or a homologue thereof and Dazl or a homologue thereof, are strongly transcribed in this cell;

(3) the cell strongly expresses the protein encoded by VASA (or CVH) or a homologue thereof;

(4) the cell does not contribute to somatic tissues when injected into a Stage X (EG&K) or a Stage 12-1 7 (H&H) recipient embryo; and/or

(5) the cells transmits the PGC genotype through the germline when injected into

Stage X (EG&K) or Stage 12-1 7 (H&H) embryos (Tajima et al. (1 993) Theriogenology 40, 509-519; Naito et al., (1 994) MoL Reprod. Dev., 39, 1 53-1 61 ; Naito et al., (1999) J Reprod. Fert. 1 1 7, 291 -298). To confirm the PGC properties of a cell, each single criterion out of these five criteria (1 ) to (5) may be sufficient, however, a combination of two out of the five criteria (1 ) to (5) is preferred, a combination of three out of the five criteria (1 ) to (5) is more preferred, a combination of four out of the five criteria (1 ) to (5) is even more preferred, and a combination of all five criteria (1 ) to (5) is particularly preferred to confirm the PGC properties of a cell.

Embryo stages are referred to herein in Roman numerals regarding the table of Eyal-Giladi and Kochav (EG&K) ((Eyal-Giladi H. and Kochav S. From cleavage to primitive streak formation: A complementary normal table and a new look at the first stages of the development of the chick. Developmental Biology, 1 976,49: 321 -327), whereby the EG&K table is concerned with the pre-laying stages of embryonic development. Embryo stages are referred to herein in Arabic numerals regarding the table of Hamburger and Hamilton (H&H) (Hamburger & Hamilton, 1 951 , A series of normal stages in the development of chick embryo. J. Morpho! 88: 49-92), whereby the H&H table is the standard for describing post-laying stages of embryonic development. There is thus some overlap between the two tables, stages X - XI (EG&K) roughly corresponding to stage 1 (H&H). In birds, primordial germ cells first appear at stage X in the ventral surface of the area pellucida, subsequently translocate from the epiblast to the hypoblast, and then migrate to an extra-embryonic region, referred to as the germinal crescent (Swift, C.H. Origin and early history of the primordial germ cells in the chick. American Journal of Anatomy, 1 914, 1 5:483- 51 6; Ginsburg M. and Eyal-Giladi H. Temporal and spatial aspects of the gradual migration of primordial germ cells from the epiblast into the germinal crescent in the avian embryo. Journal of Embryo Experimental Morphology, 1986, 95: 53-71 ). In particular, avian PGCs separate from hypoblasts around stage 4 and become located in the lacuna between hypoblasts and hyperblasts. Around stage 1 1 , PGCs become located inside the blood vessels forming in the germinal crescent region, and begin to circulate throughout the embryonic disk in the blood (initial phase of PGC-circulation). Thus, with the development of the gonadal ridge, the avian PGCs enter into the vascular system around stage 1 1 , whereby mitosis of PGCs is observed throughout the PGC-circulation phase, which continues until around stage 1 6, and wherein PGCs passively migrate to the vicinity of the gonadal ridge, where they leave the blood circulation and migrate into the gonadal anlage, i.e. the developing embryonic gonad, which later develops into testis or ovary (Kuwana T. Migration of avian primordial germ cells toward the gonadal anlage. Dev Growth Diff 1 993; 35:237-243).

Usually, the earliest identification of PGCs by morphological criteria is possible approximately 8 hours after the beginning of incubation, i.e. at stage 4 (H&H). The primordial germ cells reside in the germinal crescent from stage 4 (H&H) until they migrate through the vasculature during stage 12-1 7 (H&H). At this time, the primordial germ cells are a small population of about 200 cells. From the vasculature, the primordial germ cells migrate into the genital ridge and are incorporated into the ovary or testes as the gonad differentiates (Swift, 1 914, Am. J. Anat. 1 5, 483 - 51 6; Meyer, (1 964) Dev Biol. 1 0,1 54-190; Fujimoto et al. (1 976) Anat. Rec. 1 85,139 - 1 54). According to the present invention, the primordial germ cells are derived from germinal crescent tissue isolated from an avian embryo at stage 4 to 1 1 , preferably at stage 4 to 10, more preferably at stage 6 to 10, even more preferably at stage 7 to 10 (embryo stages in Arabic numerals herein are according to H&H, as described above). At this early stage of avian embryonic development, the PGCs are located within the germinal crescent as described above. Thus, according to the present invention, the primordial germ cells are derived from germinal crescent tissue isolated from its natural environment, i.e. an avian embryo, at an early stage, in particular prior to the migration of the PGCs in the blood and prior to entering the developing embryonic gonad. The avian embryo, from which the germinal crescent tissue is isolated, may be a male or a female embryo. Preferably, the avian embryo is a male avian embryo.

In particular, the method for establishing a long-term culture of avian primordial germ cells according to the present invention preferably comprises a step of isolating germinal crescent tissue from an avian embryo at stage 4 to 1 1 , preferably at stage 4 to 10, more preferably at stage 6 to 1 0, even more preferably at stage 7 to 10. The germinal crescent tissue from an avian embryo may be obtained by various methods known to the skilled person. However, it is preferred that freshly laid eggs are collected, preferably within one hour after the egg is laid, more preferably within 30 min after the egg is laid, even more preferably within 10 min after the egg is laid, and particularly preferably immediately after the egg is laid. Thereafter, preferably immediately after collecting, the eggs are incubated, e.g. in an incubator. The incubation may be preferably between 35°C and 40°C, more preferably between 36°C and 39°C and even more preferably between 37°C and 38°C. In particular, the incubation conditions may be chosen dependent on the species, as known by the person skilled in the art. A non-limiting example for incubation conditions, e.g. for eggs of houbara bustard, includes a temperature of 37.8°C, at a rocking angle of between 40° to 50°, with 25 - 45 % humidity. The embryos are preferably staged, e.g. during incubation of the eggs, according to the chicken embryo staging systems described above. After its isolation from the embryo, the germinal crescent tissue is preferably dissociated to obtain a cell suspension, preferably a single cell suspension, of germinal crescent tissue cells. Such a cell suspension can be achieved by way of different techniques known to the person skilled in the art. For example, the tissue may be dissociated by enzymatic means, e.g. Trypsin-EDTA (e.g. 0.25 % Trypsin-EDTA) may be added to the dissected germinal crescent tissue to dissociate the tissue. Such an enzymatic reaction may then be stopped by adding culture medium, e.g. PGC culture medium. Alternatively and/or additionally to the enzymatic dissociation, the tissue may be broken mechanically, e.g. by gently pipetting. The resulting dissociated germinal crescent tissue may optionally be centrifuged and the cell pellets thereafter re-suspended in culture medium, e.g. PGC culture medium. The cell suspension may be seeded in a culture vessel, e.g. plates with wells or culture flasks, which are known to the person skilled in the art.

Preferably, the cells of the isolated germinal crescent tissue are used to establish a primary culture.

Preferably, the method for establishing a long-term culture of avian primordial germ cells according to the present invention comprises the following steps:

a) Isolating germinal crescent tissue from an avian embryo at stage 4 to 1 1 , preferably at stage 4 to 10, more preferably at stage 6 to 10, even more preferably at stage 7 to 10; b) Establishing of a primary culture by culturing cells of the germinal crescent tissue on a feeder matrix;

c) Optionally co-culturing the primordial germ cells and somatic cells derived from germinal crescent tissue on a feeder matrix from passage 1 , preferably until passages 3 to 6, more preferably until passages 4 or 5; and

d) transferring the primordial germ cells to a culture largely devoid of, preferably without, somatic cells derived from germinal crescent tissue. In this preferred method, cells of the germinal crescent tissue isolated in step a) are used to establish a primary culture. To this end, the germinal crescent tissue is preferably dissociated, after its isolation from the embryo, in order to obtain a cell suspension, in particular a single cell suspension, of germinal crescent tissue cells. Such a cell suspension can be achieved as described above.

To establish a primary culture in step b), the cells are usually seeded into an appropriate culture vessel, e.g. a culture plate with wells, a culture flask etc.. As outlined above, the term "primary culture" (or passage 0) as used herein refers to the first culture following the isolation of cells from tissue, i.e. the first time that the cells are grown under controlled conditions, generally outside of their natural environment. In particular, in the primary culture the cells are cultured under culture conditions as described herein, on a feeder matrix as described herein, preferably on a layer of feeder cells as described herein, e.g. mouse fibroblasts, for example STO cells, and/or buffalo rat liver (B L) cells, whereby preferred feeder cells are BRL (buffalo rat liver) cells. In the primary culture, the medium may preferably be changed one or more times, e.g. a first change of medium at day 2, 3, or 4, preferably at day 3, followed by subsequent medium changes at every other day, wherein preferably one third to half of the medium is replaced in particular in the subsequent medium changes. Even when cells with typical PGC morphology, e.g. rich granula inside cytoplasm, larger in size (1 5 - 28 pm), are not observed in the germinal crescent cell suspension, they usually start appearing after a few days, e.g. after 1 , 2, 3, 4, or 5 days in culture with typical PGC morphology. In addition to PGCs, the primary culture of cells of the germinal crescent tissue may comprise somatic cells derived from the germinal crescent tissue. In particular, the somatic cells derived from the germinal crescent tissue may be isolated together with the PGCs. For example, isolated tissue samples of germinal crescent tissue may contain PGCs and somatic cells, whereby such a cell "mixture" can be used to establish a primary culture. In culture, most of the somatic cells usually attach on the feeder matrix and start proliferating soon, preferably immediately, after seeding. After the primary culture, i.e. after several days, preferably after three to nine days, more preferably after four to eight days, even more preferably after five to seven days, the cells are transferred for the first time. Preferably, the first transfer of the primordial germ cells, i.e. from passage 0 to passage 1 , is conducted after the PGC characteristics (e.g. PGC morphology) appeared and the somatic cells cover 65 to 95 %, preferably 70 to 90 %, more preferably 70 to 80 % of the feeder matrix. This transfer method, i.e. transferring the PGCs when the somatic cells cover 65 to 95 %, preferably 70 to 90 %, more preferably 70 to 80 % of the feeder matrix is also preferred for the transfers subsequent to the first transfer, preferably for every transfer until passages 3 to 6, more preferably until passages 4 or 5, even more preferably in every passage in step c).

Preferably, the method described above comprises step c). In step c) from passage 1 , preferably until passages 3 to 6, more preferably until passages 4 or 5, the primordial germ cells and somatic cells derived from germinal crescent tissue are co-cultured on a feeder matrix as described above. Such co-culturing is preferably carried out on a layer of feeder cells, e.g. mouse fibroblasts, STO cells, and/or buffalo rat liver (BRL) cells, whereby BRL (buffalo rat liver) cells are preferred, in a complete culture medium as described herein.

Alternatively, PGCs are transferred directly after the primary culture to a culture largely devoid of, preferably without, somatic cells derived from germinal crescent tissue.

If step c), i.e. a co-culture of PGCs and somatic cells is carried out, the PGCs are transferred after step c), in particular directly thereafter, to a culture largely devoid of, preferably without, somatic cells derived from germinal crescent tissue.

The term "largely devoid of" means herein, that at least 80 %, preferably at least 85 %, more preferably at least 90 %, even more preferably at least 95 %, and particularly preferably at least 98 % of the transferred cells are PGCs, in particular showing the typical PGC morphology as described above. Whether or not the method according to the invention includes a step c), the transfer of PGCs to a culture largely devoid of, preferably without, somatic cells derived from germinal crescent tissue is preferably carried out when the somatic cells derived from germinal crescent tissue grow slower and/or start to dilute. It is furthermore preferred that a transfer of the cells following step b) comprises the following sub-steps:

(i) harvesting primordial germ cells; and

(ii) harvesting the remaining cells, in particular the somatic cells derived from germinal crescent tissue.

This may apply in particular to the first transfer, i.e. directly after the primary culture ("Passage 0"), and/or to any transfer conducted in the preferred sub-step c). The term "harvesting primordial germ cells" means that as far as possible PGCs are harvested. However, due to the harvesting technique applied it may happen that a small amount of cells other than PGCs is also harvested in sub-step (i). Therefore, the term "harvesting primordial germ cells" refers to harvesting cells, wherein at least 80 %, preferably at least 85 %, more preferably at least 90 %, even more preferably at least 95 %, and particularly preferably at least 98 %, e.g. 99 % or 100 %, of the harvested cells are PGCs, in particular showing the typical PGC morphology. By "harvesting the remaining cells, in particular the somatic cells derived from germinal crescent tissue" typically harvesting of the feeder cells is largely avoided (as far as possible), thus the "remaining cells" are predominantly somatic cells derived from germinal crescent tissue. In particular, the "harvesting the remaining cells, in particular the somatic cells derived from germinal crescent tissue" refers to harvesting cells, wherein at least 80 %, preferably at least 85 %, more preferably at least 90 %, even more preferably at least 95 %, and particularly preferably at least 98 %, e.g. 99 % or 100 %, of the harvested cells are somatic cells derived from germinal crescent tissue. Preferably, after sub-step (ii) a mixture of the primordial germ cells and the somatic cells derived from germinal crescent tissue is prepared, whereby the mixture preferably comprises 10 % to 50 %, more preferably 20 % to 40 %, and even more preferably 25 % to 33 %, of the cells harvested in sub-step (ii), in particular of the somatic cells derived from germinal crescent tissue, and the mixture is seeded in a vessel containing a feeder matrix. Thus, the term "a mixture of the primordial germ cells and the somatic cells derived from germinal crescent tissue" usually refers to a mixture of the cells harvested in sub-step (i) and the cells harvested in sub-step (ii). Preferably, the mixture is a PGC-somatic cell mixed suspension. Optionally, the cells harvested in sub-step (i) and/or the cells harvested in sub-step (ii) and/or a mixture thereof may be examined, for example in respect to the cell morphology, e.g. by observation under a microscope.

It is particularly preferred that, if a step c) is performed, the above described mixture is prepared and seeded in each of the transfers of the cells in step c).

Moreover, it is preferred that in the method for establishing a long-term culture of avian primordial germ cells according to the present invention described above the avian primordial germ cells are primordial germ cells from bustard, chicken, duck, quail, hawk, or ibis, e.g. northern bald ibis ("waldrapp"), preferably primordial germ cells from bustard, in particular houbara bustard, as described above. It is also preferred that the avian primordial germ cells are primordial germ cells from an endangered bird as defined above.

In general, in the above described method for establishing a long-term culture of avian primordial germ cells according to the present invention preferably culture conditions and culture medium as described herein are used. Isolated avian PGC, culture and cell line thereof

In another aspect, the present invention provides an isolated avian primordial germ cell, which is derived from germinal crescent tissue isolated from an avian embryo at stage 4 to 1 1 , preferably at stage 4 to 10, more preferably at stage 6 to 10, even more preferably at stage 7 to 10. Such an avian PGC is particularly useful in a culture of avian primordial germ cells and/or in an avian primordial germ cell line, in particular to obtain and/or culture a culture of avian primordial germ cells and/or an avian primordial germ cell line.

Accordingly, the present invention also provides a culture of an avian primordial germ cell as described above, i.e. wherein the primordial germ cell is derived from germinal crescent tissue isolated from an avian embryo at stage 4 to 1 1 , preferably at stage 4 to 10, more preferably at stage 6 to 10, even more preferably at stage 7 to 10. As described above, the term "culture" or "cell culture" as used herein refers to cells, which are grown under controlled conditions, generally outside of their natural environment. Preferably, the cells are grown in a culture vessel, e.g. in a well of a culture plate, in a culture flask etc.. Preferably, the cells are grown under culture conditions as described herein.

The culture of avian PGCs according to the present invention preferably further comprises a feeder matrix as described herein and a culture medium as described herein. This culture medium preferably comprises the growth factors FCF₂, SCF, LIF, and IGF-1 and furthermore preferably comprises a conditioned medium as described herein, more preferably feeder cell conditioned medium, even more preferably BRL conditioned medium. Thereby, it is further preferred that the the culture medium further comprises a basal medium, preferably DMEM, more preferably knockout DMEM, animal serum, preferably FBS and/or chicken serum, an antibiotic, preferably penicillin/streptomycin, and further additives, preferably including nucleosides, a glutamine derivative, preferably GlutaMax, MEM-NEAA, a biological buffer, preferably HEPES, pyruvate (e.g. sodium pyruvate), and/or β-mercapto-ethanol. Further details of the culture medium and the culture conditions can be retrieved from the general section herein describing the culture medium and the culture conditions.

Moreover, in the culture of avian PGCs according to the present invention it is preferred that the avian primordial germ cells are cultured in vitro for at least 5 days, at least 10 days, at least 14 days, at least 25 days, at least 50 days at least 75 days, at least 100 days, at least 150 days, at least 200 days, at least 250 days, at least 300 days, and/or at least 350 days, whereby the longer the time period the more it is preferred. Furthermore, the present invention also provides a cell line of an avian primordial germ cell as described above, i.e. an avian primordial germ cell line, wherein the primordial germ cells are derived from germinal crescent tissue isolated from an avian embryo at stage 4 to 1 1 , preferably at stage 4 to 10, more preferably at stage 6 to 1 0, even more preferably at stage 7 to 10. Preferably, such a cell line is maintained under culture conditions as described herein.

The term "cell line" as used herein refers in general to a colony of cells derived and developed as a subculture from a primary culture in laboratory culture. In particular, a "cell line" is a cell culture selected for uniformity from a cell population derived from a usually homogeneous tissue source, whereby the avian PGC cell line according to the present invention is derived from germi nal crescent tissue as described above. Preferably, the cell line according to the present invention is a permanently established cell culture that will proliferate indefinitely given appropriate fresh medium and space.

Preferably, the cell line of avian primordial germ cells according to the present invention is a cell line of primordial germ cells of a male bird. Preferably, the total cell number of the cell line of avian primordial germ cells according to the present invention after three months in vitro culture is more than 1 million cells, preferably more than 1 ,5 million cells, more preferably more than 2 million cells, even more preferably more than 2,5 million cells, and particularly preferably more than 3 million cells. Moreover it is preferred that the primordial germ cell in the isolated avian primordial germ cell according to present invention, in the culture of avian primordial germ cells according to present invention, and in the cell line of avian primordial germ cells according to present invention, is preferably a bustard primordial germ cell, i.e. of the family Otididae (Bustards), more preferably a Chlamydotis (genus) primordial germ cell, and even more preferably a houbara bustard primordial germ cell, as described above. It is also preferred that the avian primordial germ cells are primordial germ cells from an endangered bird as defined above.

Preferably, the cell line of avian primordial germ cells according to the present invention is a cell line of primordial germ cells of a bustard, more preferably of a houbara bustard, even more preferably of a male houbara bustard.

Genetic Modification

An isolated avian primordial germ cell, a culture of avian primordial germ cells according to the present invention and/or a cell line of avian primordial germ cells according to the present invention as well as the methods for establishing and maintaining a culture of PGCs according to the present invention, provide an excellent system to introduce a genetic modification in avian PGCs, for example in order to introduce a genetic modification in the germ line of birds. In particular, long periods in culture are typically required in order to produce a sufficient number of cells to introduce a genetic modification by conventional electroporation or lipofection protocols. Typically, these protocols require 10^s to 10⁷ cells and therefore, production of these cells from a single precursor requires 1 7 to 24 doublings assuming that all cell divisions are (1 ) synchronous and (2) produce two viable daughter cel ls. Moreover, the introduction of a genetic modification into the genome of a cell is a usually rare event, typically occurring in one in 1 x 10⁴ to l x l O⁶ cells. Accordingly, the avian primordial germ cell in the isolated avian primordial germ cel l according to present invention, in the culture of avian primordial germ cells according to present invention, and in the cell line of avian primordial germ cells according to present invention is preferably genetically modified. The term "genetic modification" as used herein refers to the direct manipulation of an organism's genome using biotechnology. For example, DNA, in particular genes, may be inserted in the host genome, e.g. by first isolating and copying the genetic material of interest using molecular cloning methods to generate a DNA sequence, or by synthesizing the DNA, and then inserting this construct into the host organism. DNA, in particular genes, may also be removed, or "knocked out", for example using a nuclease. Furthermore, DNA , in particular genes, may also be substituted or otherwise modified. Gene targeting is a further technique, wherein homologous recombination may be used to change an endogenous gene, and which can be used to delete a gene, remove exons, add a gene, or introduce point mutations. Non-limiting examples for a genetic modification include a reporter gene, e.g. for selecting and/or observing the PGCs, a gene related to disease resistance, a gene related to a growth hormone, a gene related to a cytokine, a gene related to an interleukin, a gene related to an interferon, a gene related to an enzyme, and/or a gene related to an immunoglobulin and/or fragments thereof.

Preferably, the genetic modification in the isolated avian primordial germ cell, the culture of avian primordial germ cells or the avian primordial germ cell line according to the present invention, wherein the avian primordial germ cell is genetically modified, is the introduction of a reporter gene or of a disease resistance into an avian primordial germ cell.

In general, PGCs according to the invention, and genetically modified PGCs according to the invention in particular, may be maintained (i.e. cultured) for a long period of time in vitro prior to their introduction into recipient embryo. This long period of time allows to genetically modify said cells. According to a preferred embodiment, the cells have been in vitro cultured for at least 5 days, at least 1 0 days, at least 14 days, at least 25 days, at least 50 days at least 75 days, at least 100 days, at least 150 days, at least 200 days, at least 250 days, at least 300 days, and/or at least 350 days. Accordingly, a long-term culture of genetically modified PGCs according to the invention, in particular a long-term culture of genetically modified PGCs obtained by the method of genetically modifying avian PGCs described herein, is also preferred according to the present invention. For example, a method of genetically modifying avian PGCs according to the present invention, comprises the steps of:

a) transfecting PGCs obtained and cultured according to the methods above, with a vector;

b) selecting transfected PGCs, preferably by addition of a selection agent in the medium, such as for example antibiotics, amino-acids, hormones, etc.;

c) screening and amplification of genetically modified PGC clones, e.g. resistant PGC clones;

d) culturing said genetically modified PGCs of step c) on a feeder matrix in a culture medium as previously described.

Preferably, said culture medium of step d) is a culture medium as described herein, which comprises animal serum and preferably a combination of growth factors as described herein.

PGCs of step c) are genetically modified. Genetic modification may be performed, for example, by transient or stable transfection with the vector in the PGCs. Preferably, the PGCs are stably transfected with the vector according to techniques well known by the person skilled in the art. For example, the vector is inserted randomly into the genome of the PGCs. Preferably, the vector is inserted by homologous recombination into the genome of the PGCs.

The term "vector" as used herein refers to a natural or synthetic single or double stranded plasmid or viral nucleic acid molecule that can be transfected into cells and replicate independently of, or within, the host cell genome. A circular double stranded plasmid can be linearized by treatment with an appropriate restriction enzyme based on the nucleotide sequence of the plasmid vector. A nucleic acid can be inserted into a vector by cutting the vector with restriction enzymes and ligating the pieces together. The nucleic acid molecule can be RNA or DNA. The term "plasmid" as used herein refers to a small, circular DNA vector capable of independent replication within a bacterial or yeast host cell. Typically, the nucleic acid vector further includes at least one regulatory sequence operably linked to a nucleotide sequence coding for a "polypeptide of interest". Regulatory sequences are well recognized in the art and may be selected to ensure good expression of the linked nucleotide sequence without undue experimentation by those skilled in the art. As used herein, the term "regulatory sequences" includes promoters, enhancers, and other elements that may control expression. Standard molecular biology textbooks such as Sambrook et al. eds "Molecular Cloning: A Laboratory Manual" 2nd ed. Cold Spring Harbor Press (1 989) and Lodish et al. eds., "Molecular Cell Biology," Freeman (2000) may be consulted to design suitable expression vectors, promoters, and other expression control elements.

It should be recognized, however, that the choice of a suitable expression vector depends upon multiple factors including the choice of the host cell to be transformed and/or the type of protein to be expressed. Also useful for various applications are tissue-selective (i.e. tissue- specific) promoters, i.e., promoters from which expression occurs preferentially in cells of a particular kind of tissue, compared to one or more other types of tissue. An exemplary tissue- specific promoter is a chicken oviduct-specific promoter that is naturally associated with the proteins of avian egg whites including ovalbumin, lysozyme, ovomucoid, conalbumin and ovomucin and the like. Useful promoters also include exogenously inducible promoters. These are promoters that can be "turned on" in response to an exogenously supplied agent or stimulus, which is generally not an endogenous metabolite or cytokine. Examples include an antibiotic-inducible promoter, such as a tetracycline-inducible promoter, a heat-inducible promoter, a light-inducible promoter, or a laser inducible promoter, (e.g., Halloran et al., 2000, Development 127(9): 1 953-1960 ; Gemer et al., 2000, Int. J. Hyperthermia 1 6(2): 1 71 - 81 ; Rang and Will, 2000, Nucleic Acids Res. 28(5): 1 1205 ; Hagihara et al., 1 999, Cell Transplant. 8(4): 4314 ; Huang et al., 1 999, Mol. Med. 5(2): 129-37 ; Forster, et al., 1 999, Nucleic Acids Res. 27(2): 708-10 ; and Liu et al., 1 998, Biotechniques 24(4): 624-8, 630-2 (1998 )). As used herein the term "polypeptide of interest" or "protein of interest" refer to a polymer of amino acids of three or more amino acids in a serial array, linked through peptide bonds. The term "polypeptide" includes proteins, protein fragments, protein analogues, oligopeptides, peptides and the like. The term "polypeptide" contemplates polypeptides as defined above that are encoded by nucleic acids, produced through recombinant technology, isolated from an appropriate source or are synthesized. Non limiting examples of polypeptides are reporter polypeptides, polypeptides relating to disease resistance, growth hormones, cytokine, interleukine, interferon, enzymes, immunoglobulins or fragments thereof. The terms "transfection" or "transfected" as used herein refer to the process of inserting a nucleic acid into a host cell (i.e the avian PGC). Many techniques are well known to those skilled in the art to facilitate transfection of a nucleic acid into a prokaryotic or eukaryotic organism. These methods involve a variety of techniques including, but not limited to, treating the cells with high concentrations of salt such as, but not only, a calcium or magnesium salt, an electric field (i.e. electroporation), detergent, or liposome mediated transfection (i.e. lipofection, etc.), to render the host cell competent for the uptake of the nucleic acid molecules.

The present invention also provides a method for genetically modifying an isolated avian primordial germ cell according to the present invention, a culture of an avian primordial germ cell according to the present invention, or a cell line derived from an avian primordial germ cell according to the present invention, wherein a transposon system, preferably a PiggyBac Transposon system, is used.

Preferably, in a method for genetically modifying an isolated avian primordial germ cell as described above, a culture according to the present invention and/or an avian primordial germ cell line according the present invention a transposon system, preferably a PiggyBac Transposon system, is used. Transposon-based transgenesis is widely known to the person skilled in the art. Non-limiting examples of a transposon system, which may be used in the method for genetically modifying an isolated avian primordial germ cell as described above, include the Sleeping Beauty Transposon system and the PiggyBac Transposon system, whereby in the present invention the PiggyBac Transposon is preferred.

The PiggyBac Transposon system is well-known to those skilled in the art. Briefly, the PiggyBac Transposon system is a mobile genetic element that efficiently transposes between vectors and chromosomes via a "cut and paste" mechanism. During transposition, the PB transposase, or Super PB transposase, respectively, recognizes transposon-specific inverted terminal repeat sequences (ITRs) located on both ends of the transposon vector and moves the contents from the original sites and efficiently integrates them into TTAA chromosomal sites. The powerful activity of the PiggyBac transposon system enables genes of interest between the two ITRs in the PB vector to be easily mobilized into target genomes. The TTAA- specific transposon PiggyBac is rapidly becoming a highly useful transposon for genetic engineering of a wide variety of species.

Uses of an isolated avian PGC, a culture of avian PGC and/or an avian PGC cell line

As already mentioned above, an isolated avian primordial germ cell according to the present invention, a culture of avian primordial germ cells according to the present invention and/or a cell line of avian primordial germ cells according to the present invention can be useful in many different ways, for example for genetic modification of the avian primordial germ cell, for obtaining a germline chimera, for preservation and/or mass production of the bird, preferably of an endangered avian species as defined above, and/or for preserving the (genetic) diversity of an avian species, in particular of an endangered avian species.

Accordingly, the present invention also provides the use of an isolated avian primordial germ cell according the invention, of a culture of avian primordial germ cells according to the present invention and/or of a cell line of avian primordial germ cells according to the present invention for genetic modification of the avian primordial germ cell, in particular as described above. Thereby, the genetic modification is preferably the introduction of a reporter gene or a disease resistance into avian primordial germ cell.

Moreover, the present invention also provides the use of an isolated avian primordial germ cell according the invention, of a culture of avian primordial germ cells according to the present invention and/or of a cell line of avian primordial germ cells according to the present invention for obtaining a germline chimera. Obtaining a germline chimera according to the present invention is explained in detail below, referring to a germline chimera and/or a method for obtaining a germline chimera according to the present invention.

Furthermore, the present invention also provides the use of an isolated avian primordial germ cell according the invention, of a culture of avian primordial germ cells according to the present invention and/or of a cell line of avian primordial germ cells according to the present invention for preservation and/or mass production of the bird, preferably of an endangered avian species as defined above.

For example, a culture of avian primordial germ cells according to the present invention and/or a cell line of avian primordial germ cells according to the present invention may be used for preserving the (genetic) diversity of an endangered avian species as defined above, comprising a method for establishing a long-term culture of avian primordial germ cells according the present invention and/or a method for culturing a culture of avian primordial germ cells according to the present invention as described above. Thereby, preferably a step of cryopreservation is also comprised. Culture conditions and culture medium

In the following, the preferred culture conditions and the preferred culture medium, which can be used in the present invention in general, in particular in the method for establishing a long-term culture of avian primordial germ cells according to the present invention, for an avian primordial germ cell according to the present invention, for a culture of an avian primordial germ cell according to the present invention, as well as for a cell line derived from an avian primordial germ cell according to the present invention are disclosed. In general, the culture conditions typically comprise a "complete culture medium". The term "culture medium" in general as used herein refers to a liquid or gel designed to support the growth of cells. A "complete culture medium" refers to a basal medium, preferably a basal synthetic medium, supplemented with at least one growth factor and animal serum. Non- limiting examples of complete culture media are described in WO 03/076601 , WO 05/007840 , EP 787180, US 6,1 14,1 68, US 5,340,740, US 6,656,479, US 5,830,51 0 and in Pain et al. (1 996, Development 122:2339-2348).

As used herein, "basal medium" refers to a medium that allows, by itself, at least cell survival, and preferably, cell growth. In particular a basal medium has a classical medium formulation. Non-limiting examples of basal media include BME (Eagle's Basal Medium), MEM (minimum Eagle Medium), medium 199, DMEM (Dulbecco's modified Eagle Medium), Knockout DMEM, GMEM (Glasgow modified Eagle medium), DMEM-HamF12, Ham-F12 and Ham- F10, Iscove's Modified Dulbecco's medium, MacCoy's 5A medium, and RPMI 1640. Basal media usually comprise one or more inorganic salts (for example CaCI₂, KG, NaCl, NaHCO3, NaH₂PO4, MgSCt, etc.), one or more amino-acids, one or more vitamins (for example thiamine, riboflavin, folic acid, D-Ca pantothenate, etc.) and/or one or more other components such as for example glucose, beta-mercapto-ethanol, and sodium pyruvate. Preferably, the basal medium is a synthetic medium. A preferred basal medium according to the present invention is DMEM, more preferably Knockout DMEM, which is a basal medium optimized for growth of undifferentiated embryonic and induced pluripotent stem cells.

In the complete culture medium according to the present invention, the basal medium is supplemented with at least one growth factor and animal serum. The term "growth factor" as used herein refers to an exogenous growth factor added to the culture medium, which promotes the survival and the growth of the avian PGCs in culture. Growth factors comprise in particular cytokines and trophic factors. In the present invention such cytokines, whose action is mediated through a receptor which is associated with the gp130 protein, are preferred. Preferred cytokines include leukemia inhibitory factor (LIF), interleukin 1 1 (IL1 1 ), interleukin 6 (IL6), interleukin 6 receptor, Ciliary Neurotrophic factor (CNTF), oncostatin and cardiotrophin, which have a similar mode of action with a recruitment at the level of the receptor of a specific chain and the combination of the latter with the gp1 30 protein in monomeric or sometimes heterodimeric form. Preferred trophic factors are Stem Cell Factor (SCF), Insulin Growth factor 1 (IGF-1 ) and Fibroblast Growth Factor (FGF), more preferably basic FGF (FGF₂ or bFGF). As growth factors, a combination of FGF₂, SCF, LIF, and IGF-1 is particularly preferred.

The concentration of each of the growth factors, in particular of each of FGF₂, SCF, LIF, and IGF-1 , in the culture medium is preferably from about 0.01 to 500 ng/ml, more preferably from about 0.1 to 100 ng/ml, even more preferably from 0.5 to 50 ng/ml, and particularly preferably from 1 to 20 ng/ml. In a particularly preferred embodiment, the culture medium comprises as growth factors about 5 ng/ml for SCF and about 10 ng/ml for each of FGF₂, LIF, and IGF-1 , preferably without any further growth factors.

In the preparation of the culture medium it is preferred that the growth factors are added to the culture medium in a final step, i.e. after the other ingredients of the culture medium are added and, preferably, after the pH value is adjusted, e.g. to 7.0, for example with NaOH, e.g. 1 N NaOH and/or with HCI.

Preferably, the culture medium according to the present invention comprises animal serum. The preferred animal serum is fetal animal serum and/or chicken serum. The preferred fetal animal serum is fetal bovine serum (FBS), whereby ES cell tested FBS is in general particularly preferred. In the culture medium according to the present invention a combination of both, fetal bovine serum (preferably, ES cell tested FBS) and chicken serum, is particularly preferred. For example, the culture medium may comprise approximately from 6 to 9 % of fetal bovine serum (preferably, ES cell tested FBS) and/or from 1 to 4 % chicken serum. However, animal serum comprising serum from other animal species (e.g. horse, porcine, ungulate, etc..) may also be used. The final concentration of animal serum in the culture medium is preferably approximately from 1 to 25 %, more preferably from 5 to 20 %, even more preferably from 8% to 12 %. In a particularly preferred embodiment, the final concentration of animal serum in the culture medium is 10%. Preferably, the animal serum in the culture medium is a combination of fetal bovine serum (preferably, ES cell tested FBS) and chicken serum, whereby the percentage of chicken serum in total animal serum is preferably from 1 0 to 40 %, more preferably from 20 to 30 %, even more preferably from 22 to 28 %, e.g. 25%. Thus, the remaining part in total animal serum is fetal bovine serum, preferably ES cell tested FBS. For example, if the final culture medium contains 10 % animal serum it is particularly preferred that the final culture medium contains 7,5 % FBS (preferably, ES cell tested FBS) and 2,5 % chicken serum.

The culture medium preferably further comprises a conditioned medium. A "conditioned medium" as used herein refers to a medium in which cells, preferably feeder cells, more preferably BRL (buffalo rat liver) cells, have been cultivated already for a period of time. Preferably, the percentage of conditioned medium in the culture medium is up to 80 %, more preferably up to 70 %, even more preferable up to 60 % and particularly preferably up to 55 % of the final medium volume. In addition, it is preferred that the final culture medium contains at least 25 %, more preferably at least 33,3 %, even more preferably at least 40 %, and particularly preferably at least 45 % conditioned medium. Thus, it is particularly preferred that the culture medium comprises in its final volume 45 % to 55 % conditioned medium, more preferable 48 % to 52 % conditioned medium, e.g. 50 % conditioned medium. Alternatively, the culture medium may be based on a conditioned medium, preferably BRL conditioned medium, i.e. in this case the culture medium does not comprise any "unconditioned" basal medium.

By way of example, conditioned medium, in particular BRL conditioned medium, can be prepared according to art- recognized techniques, such as described by Smith and Hooper (1 987, Dev. Biol. 121 : 1 -9). In general, a conditioned medium is usually obtained from cells cultured for a period of time in a medium. The medium used for cultivating cells, preferably feeder cells, more preferably BRL cells, i.e. the medium which is thereafter to become the conditioned medium, is preferably a basal medium supplemented with animal serum as described above. Thereby, DMEM/10% FBS and/or Knockout DMEM/10% FBS are particularly preferred. The period of time, for which the medium is cultured with cells (e.g. feeder cells, i.e. not PGCs), i.e. the period of time for which the medium is "conditioned", is usually from 1 to 10 days, preferably from 1 to 4 days, more preferably from 2 or 3 days, even more preferably about 3 days. After such a period of time the conditioned medium is preferably collected and used. The culture of feeder cells is then supplied with fresh medium, which can again be collected after a period of time as described above. Before it is used, the conditioned medium is preferably treated by sterile filtration, e.g. through 0.22 pm filter, and optionally aliquoted and can be stored, preferably at -80°C.

A preferred conditioned medium is such a medium, which has been conditioned with the same type of feeder cells, which are preferably also used as feeder cells in the PGC culture. For example, a feeder cell conditioned medium may be obtained from a culture of feeder cells, e.g. from feeder cells cultured in basal medium supplemented with animal serum, preferably DMEM, more preferably knockout DMEM with 10% FBS. In a particularly preferred method for providing a conditioned medium, the feeder cells, preferably BRL cells, are at first seeded into a culture vessel with DMEM/10%FBS, after 2 to 4, preferably 3, days the medium is removed and replaced by knockout DMEM/10% FBS, which is collected after 2 to 4, preferably 3, days. The collected conditioned medium is preferably accumulated, filtered, e.g. through 0.22 pm filter, optionally aliquoted and stored, preferably at -80°C.

As described above, to obtain conditioned medium the preferred feeder cells are BRL (buffalo rat liver) cells, preferably BRL-3A cells, or mouse fibroblast cells, e.g. STO fibroblasts. BRL conditioned medium is particularly preferred according to the present invention. For example, BRL-3A cells are available from ATCC accession number CRL-1442. However, it is also contemplated that feeder cells comprise cells from other mammalian species (e.g; ungulate, bovine, porcine species); or avian species (e.g. Gallinacea, chicken, turkey, duck, goose, quail, pheasant) may also be used.

The culture medium of the invention may preferably comprise in addition antibiotics, such as for example penicillin and streptomycin, in particular to prevent bacterial contamination. Thereby, a combination of penicillin/streptomycin is preferred. Preferably, the concentration of antibiotic in the culture medium is from 1 to 1 000 U/ml, more preferably from 1 0 to 500 U/ml, even more preferably from 50 to 250 U/ml, and particularly preferably about 100 U/ml. For example, a combination of penicillin/streptomycin maybe used in the following concentrations of antibiotic in the final culture medium: from 1 to 1000 U/ml penicillin and from 1 to 1000 pg/ml streptomycin, more preferably from 1 0 to 500 U/ml penicillin and from 10 to 500 pg ml streptomycin, even more preferably from 50 to 250 U/ml penicillin and from 50 to 250 streptomycin, and particularly preferably about 100 U/ml penicillin and about 100 pg/ml streptomycin.

In addition, culture medium of the present invention may preferably comprise further additives, e.g. nucleosides, a glutamine derivative, preferably GlutaMax, MEM-NEAA, a biological buffer, preferably HEPES, pyruvate (e.g. sodium pyruvate), and/or β-mercapto- ethanol. In general, the further additives are typically used in concentrations according to the manufacturer.

For nucleosides, preferably the concentration in the culture medium may be for each nucleoside from 0.1 to 100 mg/l, preferably from 0.5 to 50 mg/l, more preferably from 1 to 10 mg/l. As an example "EmbryoMax" Nucleosides (Millipore) may be used, which contain, in the 100x stock solution 0.73 g/l Cytidine, 0.85 g/l Guanosine, 0.73 g/l Uridine, 0.8 g/l Adenosine, and 0.24 g/l Thymidine and which are thus preferably used in 1 /100 dilution in the culture medium.

The glutamine derivative may be for example L-glutamine or GlutaMax, whereby GlutaMax is preferred. GlutaMax is an L-alanyl-L-glutamine dipeptide, which is available for example as 200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl (100x stock solution). The glutamine derivative is preferably used in the final culture medium in a concentration from 0.1 to 100 mM, more preferably from 0.5 to 50 mM, even more preferably from 1 to 10 mM, particularly preferably about 2 mM.

MEM-NEAA, i.e. minimum-essential-medium-non-essential-amino-acids solution, is a commercially available sterile-filtered and cell culture-tested liquid formulation with Earle's Salts Base, non-essential amino acids, sodium bicarbonate (NaHCOa), and phenol red as pH indicator, but without L-Glutamine. The concentration of MEM-NEAA in the final culture medium is usually according to the manufacturer, e.g. 1 :100 in case of a 10Ox stock solution. A biological buffer serves to maintain the physiological pH. A variety of biological buffers, which are suitable for cell culture, is commercially available. Examples of biological buffers include bicarbonate buffers and HEPES, whereby in the present invention HEPES is preferred. HEPES {4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) is a zwitterionic organic chemical buffering agent, which is widely used in cell culture, largely because it is better at maintaining physiological pH despite changes in carbon dioxide concentration (produced by cellular respiration) when compared to bicarbonate buffers, which are also commonly used in cell culture. The concentration of the biological buffer, in particular HEPES, in the culture medium is preferably from 1 to 50 mM, more preferably from 5 to 30 mM, even more preferably from 8 to 12 mM, particularly preferably about 10 mM.

Preferably, the pH of the culture medium is from 6.0 to 8.0, more preferably from 6.5 to 7.5, even more preferably from 6.8 to 7.2, and particularly preferably the pH value is adjusted to 7.0. For adjustment standard techniques may be used, for example NaOH, e.g. 1 N NaOH and/or HCI may be used.

Pyruvate is an intermediary organic acid metabolite in glycolysis and the first of the Embden Myerhoff pathway that can pass readily into or out of the cell. Thus, its addition to a cell culture medium provides both an energy source and a carbon skeleton for anabolic processes. A preferred pyruvate is sodium pyruvate, which may also help to reduce fluorescent light- induced phototoxicity. Pyruvate, preferably sodium pyruvate, is preferably used in the culture medium in a concentration from 0.05 to 50 mM, more preferably from 0.1 to 10 mM, even more preferably from 0.5 to 5 mM, particularly preferably about 1 mM. Beta-mercapto-ethanol (also referred to as 2-Mercaptoethanol, β-ΜΕ or 2-ME) is assumed to act as a free radical scavenger. Beta-mercapto-ethanol is preferably used in the final culture medium in a concentration from 0.005 to 5.0 mM, more preferably from 0.01 to 1 .0 mM, even more preferably from 0.05 to 0.5 mM, particularly preferably about 0.1 mM. Thus, a particularly preferred complete culture medium according to the present invention comprises: — a basal medium, preferably DMEM, more preferably knockout DMEM, as described above;

— a conditioned medium, more preferably feeder cell conditioned medium, even more preferably BRL conditioned medium, as described above;

— animal serum, preferably FBS (preferably, ES cell tested FBS) and/or chicken serum, as described above;

— the growth factors FGF_2/ SCF, LIF, and IGF-1 , as described above;

— preferably an antibiotic, more preferably penicillin/streptomycin, as described above; and

— preferably further additives, including for example nucleosides, a glutamine derivative, preferably GlutaMax, MEM-NEAA, a biological buffer, preferably HEPES, pyruvate (e.g. sodium pyruvate), and/or β-mercapto-ethanol, as described above.

Such a culture medium is particularly suitable for maintaining (i.e. culturing) avian primordial germ cells, preferably houbara bustard primordial germ cells. This means that the culture medium is preferably suitable for culturing avian primordial germ cells according to the present invention, i.e. PGCs derived from germinal crescent tissue isolated from an avian embryo at stage 4 to 1 1 , preferably at stage 4 to 10, more preferably at stage 6 to 10, even more preferably at stage 7 to 10. However, the culture medium is preferably also suitable for maintaining avian primordial germ cells from derived from other, in particular later, embryo stages, for example chicken PGCs derived from the blood, i.e. embryo stage 12 to 1 7. Therefore, the culture medium according to the present invention can be widely used in maintaining avian primordial germ cells, and possibly additionally in maintaining other cells, preferably human/animal cells, more preferably ES cells.

Preferably, the culture medium is particularly suitable for maintaining the avian primordial germ cells in the method for establishing a long-term culture of avian primordial germ cells according to the present invention, for maintaining an avian primordial germ cell according to the present invention, for maintaining a culture of an avian primordial germ cell according to the present invention, as well as for maintaining a cell line derived from an avian primordial germ cell according to the present invention. Accordingly, the present invention provides a culture medium for maintaining avian primordial germ cells, preferably for maintaining houbara bustard primordial germ cells, comprising a basal medium, preferably DMEM, more preferably knockout DMEM; a conditioned medium, more preferably feeder cell conditioned medium, even more preferably BRL conditioned medium; animal serum, preferably FBS and/or chicken serum; the growth factors FGF₂, SCF, LIF, and IGF-1 ; preferably an antibiotic, more preferably penicillin/streptomycin; and preferably further additives, including for example nucleosides, a glutamine derivative, preferably GlutaMax, MEM-NEAA, a biological buffer, preferably HEPES, pyruvate (e.g. sodium pyruvate), and/or β-mercapto-ethanol.

Thus, the culture medium as described above is in particular sufficient for the maintenance of avian primordial germ cells, preferably houbara bustard primordial germ cells, in culture for a10 days, at least 14 days, at least 25 days, at least 50 days at least 75 days, at least 100 days, at least 150 days, at least 200 days, at least 250 days, at least 300 days, and/or at least 350 days, whereby the longer the time period, the more preferable and particularly preferable is an infinite period, if refreshed appropriately and given the cells sufficient space.

The avian PGCs according to the present invention are typically cultured on a feeder matrix, preferably on a layer of feeder cells. The feeder matrix provides the basement and produces favorable factors into the culture. The term "feeder matrix" as used herein refers to feeder cells, which may be cultured cells and/or cell lines, and/or to extracellular matrix used as such. In particular, the feeder cells could be substituted with extra-cellular matrix, for example plus bound growth factors. The feeder matrix is constructed in accordance with procedures known in the art.

Preferably, the feeder matrix comprises feeder cells, more preferably the feeder matrix is a layer of feeder cells. Thereby, it is preferred that the feeder cells are preconditioned. The term "preconditioned" as used herein means that the feeder cells are cultured in the presence of medium for a period of time prior to the depositing of cells originating from germinal crescent tissue from an avian embryo in contact with the feeder matrix, e.g. a time sufficient to initiate and establish production of auxiliary substances by the feeder matrix, for example, growth factors, nutrients or other factors. Usually a feeder matrix, preferably feeder cells, is preconditioned by culturing the feeder matrix by itself for one or more days, preferably up to five days, more preferably up to four days, even more preferably up to three days, particularly preferably two to three days, prior to the depositing of cells originating from the germinal crescent tissue from an avian embryo in contact with the feeder matrix.

Cells which can be used as feeder cells are known to the person skilled in the art. Typically, cells of the fibroblast cell type may be used as feeder cells. Preferred feeder cells are BRL (buffalo rat liver) cells, preferably BRL-3A cells (e.g. ATCC CRL-1442), and mouse fibroblast cells, e.g. STO fibroblasts. However, it is also contemplated that feeder matrices comprise cells from other mammalian species (e.g; ungulate, bovine, porcine species); or avian species (e.g. Gallinacea, chicken, turkey, duck, goose, quail, pheasant) may also be used.

Moreover, according to the present invention feeder cells may also be transfected with one or more expression vector allowing for example the constitutive expression of growth factors such as avian SCF (stem cell factor) in BRL cells. Thus, this "feeder" produces the factor in a form which is soluble and/or attached in the plasma membrane of the cells.

Preferably, the maintaining process comprises a step of establishing a monolayer of feeder cells.

Feeder cells are usually mitotically inactivated using standard techniques. For example, the feeder cells may be exposed to radiation, e.g. X or gamma radiation (e.g. 4000 Rads of gamma radiation) or may be treated with mitosis inhibitors, e.g. Mitomycin C (e.g. 10 pg/ml for 2-3 hours). Procedures for mitotically inactivating cells are usually also detailed in the information typically sent with the cells at purchase, e.g. from the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 201 10-2209. Layers, in particular monolayers, may optionally be cultured to about 80% confluency, preferably to about 90% confluency, and more preferably about 100% confluency. While configuration of the feeder cells as a layer, in particular as a monolayer, is the preferred configuration for the culture, any suitable configuration is contemplated to be within the scope of the present invention. Thus, for example, layers, mono-layers, clusters, aggregates or other associations or groupings of feeder cells are contemplated to fall within the scope of the present invention and are particularly contemplated to fall with the meaning of the term "matrix".

It is to be noted that the culture conditions, including the culture medium, as disclosed herein, are preferably applied to any avian primordial germ cell according to the present invention, including the cells of the isolated germinal crescent tissue, which are also preferably maintained under the above described conditions.

Since the culture conditions as described herein are in general useful for maintaining (i.e. culturing) an avian primordial germ cell according to the present invention, a culture of an avian primordial germ cell according to the present invention, and a cell line derived from an avian primordial germ cell according to the present invention, the present invention provides, in another aspect, a method for maintaining an isolated avian primordial germ cell according to the present invention, a culture of an avian primordial germ cell according to the present invention, or a cell line derived from an avian primordial germ cell according to the present invention, wherein the avian primordial germ cells are maintained in a culture medium on a feeder matrix, the culture medium comprising the growth factors FGF₂, SCF, LIF, and IGF-1 and preferably a conditioned medium, more preferably feeder cell conditioned medium, even more preferably BRL conditioned medium.

In this method for maintaining an isolated avian primordial germ cell according to the present invention, a culture of an avian primordial germ cell according to the present invention, or a cell line derived from an avian primordial germ cell according to the present invention, the PGCs are preferably maintained for at least 10 days, at least 14 days, at least 25 days, at least 50 days at least 75 days, at least 100 days, at least 1 50 days, at least 200 days, at least 250 days, at least 300 days, and/or at least 350 days, whereby the longer the time period, the more preferable and particularly preferable is an infinite period, if refreshed appropriately and given the cells sufficient space. In particular, the culture medium and the feeder matrix as used herein are a culture medium and a feeder matrix as described above, for example a complete culture medium as defined above, whereby the complete culture medium preferably comprises a conditioned medium, preferably B L conditioned medium as described above.

Preferably, the concentration of each of the growth factors, in particular of each of FGF₂, SCF, LIF, and IGF-1 , in the culture medium is comprised between about 0.01 to 1 00 ng/ml, preferably, 0.1 to 50 ng/ml, more preferably 1 to 20 ng/ml, and even more preferably about 5 ng/ml for SCF and about 10 ng/ml for each of FGF₂, LIF, and IGF-1 .

More preferably, in the method for maintaining an isolated avian primordial germ cell according to the present invention, a culture of an avian primordial germ cell according to the present invention, or a cell line derived from an avian primordial germ cell according to the present invention, the avian primordial germ cells are maintained in a preferred culture medium as described above. This means, that the culture medium comprises:

— a basal medium, preferably DMEM, more preferably knockout DMEM, as described above;

— a conditioned medium, preferably feeder cell conditioned medium, more preferably BRL conditioned medium, as described above;

— the growth factors FGF₂, SCF, LIF, and IGF-1 , as described above;

Preferably, the method for maintaining an isolated avian primordial germ cell according to the present invention, a culture of an avian primordial germ cell according to the present invention, or a cell line derived from an avian primordial germ cell according to the present invention includes a step of transferring the cells, i.e. placing the cells from one culture vessel into another culture vessel. Thereby, the cells undergo a subculture (passage) process as described above. By subculturing (passage) the cells are kept at a sufficiently low density to stimulate further growth. Preferably, in a transfer the cells are dissociated mechanically and/or enzymatically. Mechanical dissociation can be achieved, for example, by gently pipetting the cells. Enzymatic dissociation can be achieved, for example, by treatment with Trypsin-EDTA, e.g. 0.25 % Trypsin-EDTA.

Preferably, in the method for maintaining an isolated avian primordial germ cell according to the present invention, a culture of an avian primordial germ cell according to the present invention, or a cell line derived from an avian primordial germ cell according to the present invention the cells are transferred every three to nine days, more preferably every four to eight days, and even more preferably every five to seven days.

Moreover, it is preferred that the culture medium in the method for maintaining an isolated avian primordial germ cell, a culture of an avian primordial germ cell, or a cell line derived from an avian primordial germ cell further comprises DMSO (dimethyl sulfoxide), in particular 10% DMSO, and that the method further comprises a step of freezing a culture of avian primordial germ cells.

Such freezing may be useful in cryopreservation, e.g. if a culture of avian primordial germ cells according to the present invention and/or a cell line of avian primordial germ cells according to the present invention is used for preserving the (genetic) diversity of an endangered avian species as defined above.

In yet another aspect, the present invention provides a method for culturing a culture of avian primordial germ cells, wherein the avian primordial germ cells are maintained in a culture medium on a feeder matrix, the culture medium comprising the growth factors FGF₂, SCF, LIF, and IGF-1 and preferably the culture medium comprises conditioned medium as described above.

Preferably, in the method for culturing a culture of avian primordial germ cells according to the present invention, the culture of avian primordial germ cells is obtained by a method for establishing a long-term culture of avian primordial germ cells as described above. Moreover, it is preferred that in the method for maintaining an isolated avian primordial germ cell according to the present invention, a culture of an avian primordial germ cell according to the present invention, or a cell line derived from an avian primordial germ cell according to the present invention as described above and in the method for culturing a culture of avian primordial germ cells according to the present invention as described above the avian primordial germ cells are primordial germ cells from bustard, chicken, duck, quail, hawk, or ibis, e.g. northern bald ibis ("waldrapp"), preferably primordial germ cells from bustard, in particular houbara bustard, as described above. It is also preferred that the avian primordial germ cells are primordial germ cells from an endangered bird as defined above.

Molecular analysis of avian PGCs

In a further aspect, the present invention also provides a method for molecular analysis of an avian primordial germ cell, in particular of a houbara bustard primordial germ cell, a primer pair for such molecular analysis and a respective oligonucleotide.

In general, molecular analysis of cultured avian primordial germ cells provides an important tool to confirm that the cultured cells have the properties of primordial germ cells, e.g. in contrast to embryonic germ (EG) cells. Typically, such molecular analysis thus refers to the expression profile of germ cell specific genes. Such analysis is known in the art, for example from WO 2006/084035 for chicken PGCs.

Examples of germ cell specific genes include DAZL and VASA (or the chicken VASA homologue CVH oi the Drosophila gene VASA, respectively). DAZL codes for "Deleted in azoospermia-like" protein, which is expressed in prenatal and postnatal germ cells of males and females, and mutations in DAZL were linked to severe spermatogenic failure and infertility in males. VASA is essential for germ cell development and was first identified in Drosophila melanogaster, where VASA expression is seen in germ cells, specifically the germline stem cells (GSC's) of female ovaries and in the early stages of spermatogensis in the male testis. Similarly, its chicken homologue CVH is known to be a germline specific gene, which is restricted to cells within the germline of chickens and is expressed by approximately 200 cells in the germinal crescent (Tsunekawa N, Naito M, Sakai Y, Nishida T and Noce T. Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells. Development, 127: 2741 -2750. 2000).

Preferably, the molecular analysis according to the present invention may additionally refer to the expression profile of pluripotency related genes, since some stem cell related genes are known to be expressed in PGCs. Therefore, molecular analysis of the expression profile of germ cell specific genes and of the expression profile of pluripotency related genes may be combined.

Examples of pluripotency related genes include /V C (also referred to as c-Myc), the Oct4 homologues POUVand NANOG( . Lavial F et al., 2007, Development. 1 34(1 9):3549-63), AZ/^Kruppel-like factor 4), and SOX2.

Since the houbara genomic sequence is not available yet, the present invention provides a method for molecular analysis of an avian primordial germ cell, in particular a method for molecular analysis of a Houbara bustard primordial germ cell, wherein an oligonucleotide is used, in particular as primer and/or probe, said oligonucleotide comprising or consisting of a sequence according to any of SEQ ID NO: 1 to SEQ ID NO: 6 or sequence variants thereof (cf. Table 1 ).

Table 1 :

Additionally, the present invention also provides said oligonucleotide, an oligonucleotide, in particular an oligonucleotide, comprising or consisting of a sequence selected from the group consisting of: a sequence according to SEQ ID NO: 1 or sequence variants thereof, a sequence according to SEQ ID NO: 2 or sequence variants thereof, a sequence according to SEQ ID NO: 3 or sequence variants thereof, a sequence according to SEQ ID NO: 4 or sequence variants thereof, a sequence according to SEQ ID NO: 5 or sequence variants thereof and a sequence according to SEQ ID NO: 6 or sequence variants thereof. Furthermore, the present invention also provides a primer pair for molecular analysis of an avian primordial germ cell, in particular of a houbara bustard primordial germ cell, the primer pair comprising at least one oligonucleotide as defined above.

As used herein, the term "oligonucleotide" refers to 6 to 50 nucleotides in length, preferably to 8 to 35 nucleotides in length. A "sequence variant" as used herein refers to any alteration in a reference sequence, whereby a reference sequence is in particular any of the sequences according to SEQ ID NO: 1 to SEQ ID NO: 6. In particular, a "sequence variant" has an altered sequence in which one or more of the nucleotides in the reference sequence is deleted, or substituted, or one or more nucleotides are inserted into the sequence of the reference nucleotide sequence. Nucleotides are referred to herein by the standard one-letter designation (A, C, G, or T). Preferred sequence variants share at least 80%, preferably at least 90 %, more preferably at least 90 %, even more preferably at least 95% and particularly preferably at least 98% sequence identity compared to the reference sequence. As used herein, the term "% sequence identity", has to be understood as follows: Two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment. A % identity may then be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length. In the above context, a sequence having a "sequence identity" of at least, for example, 95% to a reference sequence, is intended to mean that the subject sequence is identical to the reference sequence (i.e. any sequence according to SEQ ID NO: 1 to SEQ ID NO: 6) except that the subject sequence may include up to five nucleotide alterations per each 100 nucleotides of the reference sequence. In other words, to obtain a sequence of at least 95% identity to a reference sequence, up to 5% (5 of 100) of the nucleotides in the subject sequence may be inserted or substituted with another nucleotide or deleted. Methods for comparing the identity and homology of two or more sequences are well known in the art. The percentage to which two sequences are identical can for example be determined by using a mathematical algorithm. A preferred, but not limiting, example of a mathematical algorithm which can be used is the algorithm of Karlin eta/. (1993), PNAS USA, 90:5873-5877. Such an algorithm is integrated in the BLAST family of programs, e.g. BLAST or N BLAST program (see also Altschul et a/., 1 990, J. Mol. Biol. 21 5, 403-410 or Altschul et al. (1 997), Nucleic Acids Res, 25:3389-3402), accessible through the home page of the NCBI at world wide web site ncbi.nlm.nih.gov) and FASTA (Pearson (1 990), Methods Enzymol. 183, 63-98; Pearson and Lipman (1988), Proc. Natl. Acad. Sci. U. S. A 85, 2444-2448.). Sequences which are identical to other sequences to a certain extent can be identified by these programmes.

Preferably, the method for molecular analysis of an avian primordial germ cell, in particular of a Houbara bustard primordial germ cell, according to the present invention comprises a step, wherein a nucleic acid of the avian primordial germ cell is amplified. The amplification is preferably performed by polymerase chain reaction (PCR) or reverse-transciptase PCR (RT- PCR).

A preferred method for molecular analysis of an avian primordial germ cell, in particular of a Houbara bustard primordial germ cell, according to the present invention comprises a step, wherein a nucleic acid of the avian primordial germ cell is amplified using a primer pair selected from the group consisting of:

1 ) an oligonucleotide comprising or consisting of a sequence according to SEQ ID NO:

1 or sequence variants thereof and an oligonucleotide comprising or consisting of a sequence according to SEQ ID NO: 2 or sequence variants thereof;

2) an oligonucleotide comprising or consisting of a sequence according to SEQ ID NO:

3 or sequence variants thereof and an oligonucleotide comprising or consisting of a sequence according to SEQ ID NO: 4 or sequence variants thereof; and

3) an oligonucleotide comprising or consisting of a sequence according to SEQ ID NO:

5 or sequence variants thereof and an oligonucleotide comprising or consisting of a sequence according to SEQ ID NO: 6 or sequence variants thereof. Cermline chimera and method for obtaining a germ line chimera and method for obtaining a pure species offspring from an interspecies germline chimera

The present invention further provides a method for obtaining an interspecies or intraspecies germline chimera, comprising the following steps:

(i) Establishing a long-term culture of avian primordial germ cells according to the present invention or maintaining an isolated avian primordial germ cell according to the present invention, a culture of an avian primordial germ cell according to the present invention, or a cell line derived from an avian primordial germ cell according to the present invention; and

(ii) Providing an interspecies or intraspecies germline chimera by introducing the primordial germ cells of step (i) into a recipient of the same or different avian species.

In particular, the recipient in step (ii) is an embryo, in particular an avian embryo. In particular, the primordial germ cells of step (i) are primordial germ cells, which have been cultured in vitro.

The term "germline chimera" as used herein refers to an organism having germ cells, which are not genetically identical to the organisms own cells. In an "interspecies germline chimera", the germ cells, which are not genetically identical to the organisms own cells, are derived from a different avian species than the species of the recipient. In an "intraspecies germline chimera", in contrast, the recipient is of the same avian species as the germ cells, which are not genetically identical to the organisms own cells. However, an "intraspecies germline chimera" includes for example a chimera having germ cells from a different subspecies, race etc. as compared to the organism's own cells. Importantly, in the germline chimera the cells, which are not genetically identical to the organisms own cells, contribute exclusively to the germ cells. Thus, typically no phenotypic chimera are observed.

Since the production of a germline chimera typically requires at least 10³ cells, the culture conditions must be sufficiently robust to allow at least the cells to grow to this extend and to maintain their PGC properties as outlined above. In particular, an isolated avian primordial germ cell according to the present invention, a culture of avian primordial germ cells according to the present invention and/or a cell line of avian primordial germ cells according to the present invention as well as the methods for establishing and maintaining a culture of PGCs according to the present invention, provide an excellent system enabling that the length of the PGC culture can be extended while the genotype and phenotype of the cells as true PGCs is preserved. Such PGCs can be introduced into recipient embryos for example at a point in embryonic development when the germline competent cells are migrating to the gonad. Due to their nature as true PGCs, the introduced PGCs then contribute exclusively to the nascent population of spermatogonia or oogonia (i.e., the precursors of sperm and eggs) in the resulting animals, i.e. the "germline chimera". In such a resulting animal, the entirety of the somatic tissue is thus derived from the recipient embryo, whereas the germline contains contributions from the introduced PGCs, i.e. from the "donor cells", typically in addition to germline cells of the recipient embryo. If both, the introduced PGCs and the recipient embryo's cells, contribute to the germline, the offspring of such germline chimeras is derived either from the donor cell, i.e. the introduced PGC, or from the recipient embryo.

Preferably, in the method for obtaining a germline chimera according to the present invention, the primordial germ cells of step (i) are cultured in vitro iox at least 5 days, at least 1 0 days, at least 14 days, at least 25 days, at least 50 days at least 75 days, at least 1 00 days, at least 1 50 days, at least 200 days, at least 250 days, at least 300 days, and/or at least 350 days or even longer, before they are introduced into a recipient in step (ii). Preferably, at least 200 cultured primordial germ cells of step (i), at least 500 cultured primordial germ cells of step (i), at least 1000 cultured primordial germ cells of step (i), at least 2000 cultured primordial germ cells of step (i), at least 4000 cultured primordial germ cells of step (i), at least 5000 cultured primordial germ cells of step (i) are introduced into a recipient in step (ii).

The cultured primordial germ cells introduced into the recipient avian embryo may be a mixed population of female and male primordial germ cells. However, the method for obtaining a germline chimera according to the present invention may comprise the additional step of determining the sex of recipient embryo prior the introduction of the cultured primordial germ cells. Preferably, the recipient and/or the cultured primordial germ cells are previously sexed before introduction. Preferably, female cultured primordial germ cells are introduced into a female recipient and male cultured primordial germ cells are introduced into a male recipient.

Moreover, it is preferred in the method for obtaining a germline chimera according to the present invention that the cultured primordial germ cells of step (i) are genetically modified, in particular as described above. For example, a genetic modification relating to a reporter gene, e.g. GFP (green fluorescent protein), and/or to a gene relating to a disease resistance may be particularly useful in the cultured primordial germ cells of step (i) in the method for obtaining a germline chimera according to the present invention. A reporter gene may be used for selecting embryos, which are germline chimeras, and observing the development of the introduced PGCs.

Preferably, in the method for obtaining a germline chimera according to the present invention, the primordial germ cells of step (i) are introduced into the sub-germinal cavity of the recipient avian embryo or in the dorsal aorta of the recipient avian embryo, whereby the introduction of the cultured primordial germ cells of step (i) into the sub-germinal cavity of the recipient avian embryo is more preferred. The term "subgerminal cavity" refers to the space between the blastoderm and the yolk. This space is created when the blastoderm cells absorb fluid from the albumin and secrete it between themselves and the yolk.

Preferably, in the method for obtaining a germline chimera according to the present invention, the cultured primordial germ cells of step (i) are introduced into a recipient avian embryo at a stage from stage X (EG&K) to stage 1 7 (H&H). More preferably, the recipient avian embryo is at stage X or at a stage from stage 14 to stage 1 7, even more preferably at a stage from stage 1 5 to stage 1 6. In particular, when introduced at stage X, introduction into the sub-germinal cavity of the recipient avian embryo is preferred, whereas introduction into the dorsal aorta of the recipient avian embryo is preferred, when introduced at a stage from stage 1 4 to stage 1 7, more preferably at a stage from stage 1 5 to stage 1 6. Furthermore, in the method for obtaining a germline chimera according to the present invention the recipient embryo may derive from a freshly laid un-incubated egg or from an incubated embryo. In a preferred embodiment of the method for obtaining a germline chimera according to the present invention, the recipient embryo derives from a freshly laid un- incubated egg and comprises between around 5000 to around 70000 cells.

The donor of the avian primordial germ cells, which are cultured in step (i) of the method for obtaining a germline chimera according to the present invention may be any bird. However, preferably the donor of the avian primordial germ cells is an endangered bird as defined above. Alternatively, or additionally, it is preferred that the donor of the avian primordial germ cells is of the order Gruiformes, preferably of the family Otididae (Bustards), more preferably of the genus Chlamydotis, and even more preferably the donor of the avian primordial germ cells is a houbara bustard {Chlamydotis undulata).

The recipient of the avian primordial germ cells, which are cultured in step (i) of the method for obtaining a germline chimera according to the present invention may be any bird. Preferably, the recipient of the avian primordial germ cells is a chicken or a bird of the order Gruiformes. If the recipient of the avian primordial germ cells is a chicken, a chicken of White Leghorn strain is particularly preferred. If the recipient of the avian primordial germ cells is of the order Gruiformes, the family Otididae (Bustards) is preferred, genus Chlamydotis is more preferred and a houbara bustard (Chlamydotis undulata) is even more preferred. In a further aspect, the present invention also provides a germline chimera, having a germline comprising a primordial germ cell derived from a culture of avian PGCs according to the present invention or from an avian primordial germ cell line according to the present invention. Thus, in a germline chimera according to the present invention, the primordial germ cells derived from a culture according to the present invention or from an avian primordial germ cell line according to the present invention are typically located in the gonads.

Preferably, the germline chimera according to the present invention comprises a primordial germ cell as defined above, which is derived from an endangered bird as defined above, and/or from a bird of the order Gruiformes, preferably of the family Otididae (Bustards), more preferably of the genus Chlamydotis, and even more preferably the primordial germ cell as defined above is derived from a houbara bustard {Chlamydotis undulata). Moreover, it is preferred that the germline chimera according to the present invention is obtainable, preferably is obtained by, a method for obtaining a germline chimera according to the present invention as described above, in particular also regarding the preferred embodiments of this method as outlined above.

In a further aspects, the present invention also provides a method for obtaining a pure species offspring from an interspecies germline chimera comprising the following steps:

(i) Obtaining an interspecies germline chimera by a method according to any of claims 37 to 41 ;

(ii) Cross-breeding the interspecies germline chimera with

a. a pure species bird, which is of the same avian species as the donor of the primordial germ cells introduced in the interspecies germline chimera in step (i); or

b. an interspecies germline chimera obtained by a method according to any of claims 37 to 41 , wherein the species of the donor of the introduced avian primordial germ cells and the species of the recipient embryo correspond to the species of the donor of the introduced avian primordial germ cells and the species of the recipient embryo of the interspecies germline chimera obtained in step (i); and

(iii) selecting pure species offspring, which is preferably of the same avian species as the donor of the primordial germ cells introduced in the interspecies germline chimera in step (i). Thereby, a„pure species offspring" refers to an offspring, which has cells of only one single avian species. Thus, the offspring of an interspecies germline chimera, which is to be obtained in this method, in particular which is selected in step (iii) is not an interspecies chimera.

For cross-breeding in step (ii) preferably fertile birds of opposite sex are used. Thereby, the interspecies germline chimera obtained in step (i) may be either crossed with a pure species bird, which is of the same avian species as the donor of the primordial germ cells introduced in the interspecies germline chimera in step (i). In a preferred embodiment, the pure species bird is a wild-type bird. For example, if in step (i) primordial germ cells of a houbara bustard were introduced into a chicken embryo, the resulting interspecies germline chimera is preferably crossed with a pure species houbara bustard, which is preferably wild-type. Alternatively, in step (ii) the interspecies germline chimera obtained in step (i) may also be crossed with an interspecies germline chimera obtained by a method for obtaining an interspecies germline chimera as described herein, wherein the species of the donor of the introduced avian primordial germ cells and the species of the recipient embryo correspond to the species of the donor of the introduced avian primordial germ cells and the species of the recipient embryo of the interspecies germline chimera obtained in step (i). For example, if in the interspecies germline chimera obtained in step 1 the donor of the primordial germ cells is a houbara bustard and the recipient is a chicken, then such an interspecies germline chimera may be crossed with an interspecies germline chimera, wherein also the donor of the primordial germ cells is a houbara bustard and the recipient is a chicken.

The offspring selected in step (iii) is preferably F1 progeny to the interspecies germline chimera obtained in step (i). Preferably, the pure species offspring selected in step (iii) is of the same avian species as the donor of the primordial germ cells introduced in the interspecies germline chimera in step (i), e.g. if the introduced primordial germ cells are houbara bustard, the pure species offspring selected in step (iii) is preferably also houbara bustard.

Usually, the person skilled in the art is familiar with the methodology of cross-breeding and selecting the offspring. However, a more detailed description of cross-breeding an interspecies germline chimera with a pure species bird, which is of the same avian species as the donor of the primordial germ cells, (step ii) a.) and of selecting pure species offspring is provided in Wernery U. et al., 2010, PLoS One. 2010 Dec 29;5(12):e1 5824, which is hereby incorporated by reference, in particular regarding the molecular analysis of chimeric embryos and of semen samples from chimeric adults, the progeny test and the species identification and parentage test of the resulting progenies.

Preferably, in the method for obtaining a pure species offspring from an interspecies germline chimera as described above, the pure species offspring is an endangered bird and/or a bird of the order Gruiformes, preferably of the family Otididae (Bustards), more preferably of the genus Chlamydotis and even more preferably a houbara bustard {Chlamydotis undu/ate).

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications fall within the scope of the appended claims. All references cited herein are herewith incorporated by reference.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE FIGURES

In the following a brief description of the appended figures will be given. The figures are intended to illustrate the present invention in more detail. However, they are not intended to limit the subject matter of the invention in any way.

Figure 1 shows for Example 3 the collection of germinal crescent (circled area) containing PGCs from Houbara Bustard embryo (stage 10).

Figure 2 shows for Example 4 primary cultures of Houbara Bustard germinal crescent cells. (A) Freshly seeded Houbara Bustard germinal crescent cells. (B) Houbara PGCs appeared after three to four days culture. (C) Single PGCs after seven days culture. (D) Cluster of Houbara Bustard PGCs after seven days culture. shows for Example 5 derivation cultures of Houbara Bustard germinal crescent PGCs. (A) Houbara PGCs in single cell or chain form losing granules after 10 days culture. (B) Houbara PGCs clumps attached and losing granules after 10 days culture. (C) Female Houbara PGCs derived clump tightly compacted and attached on feeders, losing granules and stopped proliferation after 12 days culture. (D) Female PGCs clumps with granules in cytoplasm kept proliferating after 20 days culture. (E) Houbara PGCs in single cells with granules, and short PGCs chain losing granules. (F) PGCs single cells in culture. (G) Houbara PGCs clumps attached on BRL feeders. (H) Actively mitotic male Houbara Bustard PGCs on BRL feeders in culture after 35 days culture. shows for Example 6 maintenance cultures of Houbara Bustard PGCs. (A) Newly seeded Houbara single PGCs. (B) Houbara PGCs on the 5^th day culture of the passage (clumps). shows for Example 7 a culture of Houbara Bustard cPGCs from embryonic blood source (stage 14). (A) Migratory Houbara Bustards cPGCs among embryonic blood cells at stage 1 4. (B) cPGCs derived clump after 3 days culture. (C) and (D) Formation of chain or shish kabab shape of dividing Houbara Bustard cPGCs after 1 0 days culture. (E) and (F) Houbara Bustard cPGCs stopped proliferation after 1 3 days culture. shows for Example 8 the karyotype of cultured Houbara Bustards PGCs. shows for Example 9 the detection of the gene expression profile of long-term cultured Houbara Bustard PGCs. shows for Example 10 immunofluorescence stain of Houbara PGCs with anti- CVH. (A) negative control chicken PGCs. (B) negative control Houbara PGCs. (C) positive control chicken PGCs with anti-CVH. (D) positively stained Houbara PGCs with anti-CVH. shows for Example 1 1 transfection of cultured Houbara PGCs with Piggybac transposon system carrying GFP gene. (A) Houbara PGCs grow in culture in light field. (B) Houbara PGCs grow in culture in fluorescent field. (C) Dispersed Houbara PGCs suspension in light field. (D) Dispersed Houbara PGCs suspension in fluorescent field. shows for Example 12 migration of Houbara green PGCs in chicken embryos. (A) Houbara PGCs in chicken embryos at 24, 42 and 72 hours after being injected into blastoderm. (B) and (C) Testis of chicken embryo (8 dpi) containing Houbara PGCs. shows for Example 13 exogenous green Houbara PGCs derived germ cells in the testis of 21 day old Houbara embryo. (A) Overview of Houbara Bustard testis under bright field. (B) Overview of Houbara Bustard testis under fluorescence field (to show the green Houbara PGCs derived germ cells). (C) The green Houabra PGCs derived germ cells in higher magnification. shows for Example 13 exogenous male green Houbara PGCs derived germ cells in the ovary of 22 day old Houbara embryo. (A) Ovarian tissue of Houbara Bustard under fluorescence field (to show the green Houabra PGCs derived germ cells). (B) The green Houbara PGCs derived germ cells (in higher magnification). EXAMPLES

In the following, particular examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the scope of the appended claims. Example 1 : Preparation of culture medium and BRL-3A conditioned medium

Table 2 shows the formula of an example of a PGCs culture medium of the present invention, which is useful in particular for culturing Houbara Bustard PGCs. Table 2: Formula of an example of PGCs culture medium

Stock Volume (50ml*) Working cone.

concentration

Knockout DMEM 1 x 1 6.9ml

BRL condition medium 1 x 25ml 50%

ES cell tested FBS 1 x 3.75ml 7.5%

Chicken serum 1 x 1 .25ml 2.5%

Nucleosides 100x 0.5ml 1 x

Glutamax 100X 0.5ml 1 x

MEM-NEAA 1 00X 0.5ml 1 x

HEPES 100X 0.5ml 1 x

Sodium Pyruvate l OOx 0.5ml 1 x B-mercaptoethanol 50mM (500x) 0.1 ml 0.1 mM

Penici II ion/streptomycin 100x 0.5ml 1 Χ

FGF₂ 25ug/ml (2500x) 20μΙ 10ng/ml

SCF l Oug/ml (2000x) 25μΙ 5ng/ml

LIF 10ug ml (1000X) 50μΙ 10ng ml

IGF-1 50ug/ml (5000X) 5μΙ 10ng/ml

^:or preparation of 50ml culture medium

Table 3 shows the commercially available ingredients of the above PGCs culture medium with examples of the respective suppliers.

Table 3: Ingredients of the example for a PGCs culture medium with examples of suppliers

A. Preparation of culture medium:

1 . 25ml BRL- conditioned medium were taken into a 50ml tube; 10ml Knockout DMEM, and chicken serum, FBS and all the other ingredients except the growth factors were added.

2. The pH value was adjusted to 7.0 with 1 N NaOH.

3. The rest of the Knockout DMEM was added to make the total volume to be 50ml. This medium can be stored at 4°C for a few weeks.

4. To make the PGCs culture medium, the growth factors (FGF₂, SCF, LIF, IGF-1 ) were added as shown in Table 2 from the stocks (cf. Table 3) into the above medium. The prepared PGCs culture medium can be stored at 4°C for a few weeks. It is recommended to add growth factor stock to freshly prepared medium before use. B. Preparation of BRL-3A conditioned medium:

BRL-3A cells (Buffalo Rat Liver cells, purchased from ATCC, ATCC-Number: CRL-1442) were used to prepare a conditioned medium as follows.

1 . BRL-3a cells were seeded into a T75 flask with a density of 1 .1 x 10⁶ cells in 1 5-20ml DMEM (1 0% FBS).

2. After 3 days culture, the cells become confluent.

3. The medium was removed, and replaced with 20ml Knockout DMEM/10% FBS.

4. The medium was conditioned for 3 days, collected, and replaced with fresh medium, for 4 times.

5. Conditioned media were accumulated, filtered through 0.22pm filter, aliquot, and stored in -80C freezer. Condition medium can be used within one year.

Example 2: Preparation of BRL feeder cells BRL-3A feeder cells were purchased from ATCC, and cultured in DMEM/10% FBS, and passaged every three days. The cells for feeder preparation are not cultured beyond one month of total culture period, before being mitotically inactivated by X-ray irradiation or Mitomycin C treatment.

Irradiation:

1 . BRL-3a cells were maintained in T75 flask in DMEM/10% FBS, and grew into about

80% confluent.

2. The medium was removed, and cells were washed once with 20ml PBS (-).

3. Cells were treated with 2ml 0.25% Trypsin-EDTA into single cell suspension, and were harvested in DMEM/10% FBS.

4. Cells were irradiated with X-ray at the dose of 4000 rads.

5. Irradiated cells were frozen down in medium with the addition of 10% DMSO.

Mitomycin C:

1 . BRL-3A cells were cultured into 80-90% confluent in growth media in T75 flask.

2. The medium was removed, and replaced with 15ml fresh culture medium added with Mitomycin C (Sigma m0503) at a concentration of 10pg/ml.

3. Cells were returned to the incubator, and continued incubating for 2.5 hours.

4. Mitomycin C medium was removed, and cells were washed with 20ml PBS(-), three times.

5. 2ml 0.25% Typsin-EDTA was added. The single cells suspension was harvested, and frozen down in medium with addition of 1 0% DMSO.

Feeder cells seeding:

Mitotically inactivated BRL-3A cells were thawed in a 37.5°C water bath, and an equal volume of DMEM/10%FBS was added, and centrifuged at 1450rpm/ min, 4°C. The cell pellets were re-suspended in certain volume of DMEM/10% FBS. Cells were seeded in a 48 well plate (Falcon, 3230) 1 x10⁵ cells/ well, or a 24 well plate, 2x10⁵ /well. Feeder cells were cultured for two to three days before seeding the PGCs, in particular the Houbara Bustard PGCs. Usually, the feeder cells were generally cultured in DMEM/10% FBS until seeding of the PGCs. From seeding of PGCs onwards usually PGC culture medium as described herein is used as medium. Example 3: Collection of tissue from germinal crescent of a Houbara Bustard embryo

Houbara Bustard eggs were obtained from the birds which were bred in captivity over three generations in a captive breeding farm, from the third generation of parent flocks. Freshly laid Houbara Bustard egg were collected, and disinfected in F10 for 20 minutes, and incubated with the blunt end up in the incubator under 37.8°C, at a rocking angle of ±45° , with 30- 40% humidity.

The Houbara eggs were incubated for 40-48 hours. Eggs were opened from the blunt end, and a piece of egg shell was removed by forceps. A small piece of shell inner membrane was dissected and the embryo was made visible. The embryos were observed and staged parallel to chicken embryo staging system (Hamburger V, Hamilton HL. A series of normal stage in the development of the chick embryo. J Morphol 1 951 ; 88:49-92). The embryos at stage 7 (1 somite) to stage 1 0 (1 0 somites) were used for germinal crescent collection. Any embryo younger than stage 7 was sealed and put back into the incubator until it reached the desired stage.

About half of the egg white was carefully removed, and to orientate the embryo to be on top of the yolk, the egg content can be discharged into another empty container. Finally, Houbara embryos were put into a petri dish with the embryo on the top. The remaining egg white was wiped off the surface of the embryo with tissue paper.

A ring prepared from filter paper with a diameter of 2 centimetres was put around the peripheral area opaca. The whole embryo was dissected out with scissors along the outer edge of the paper ring (Figure 1 ). The whole embryo was taken out from the yolk, and put into PBS(-) with the abdomen side up in a petri dish. Yolk granular was cleaned up by gentle pipetting. The whole germinal crescent area was dissected out with the tip of a 1 ml insulin syringe. Dissected tissues were transferred into 1 .5 ml Eppendorf tubes with 20μΙ PBS(-). Remaining tissues were kept in -20°C freezer for molecular sexing use. 50μΙ 0.25% Trypsin- EDTA were added to dissociate the tissue, kept at room temperature for 2 minutes, and stopped by adding 100μ! PGC growth media, and gently pipetted to break the tissue. The mixture was centrifuged at 1450 rpm for 5 minutes at 4°C. Cell pellets were re-suspended in 300 μΙ PGCs culture medium, and seeded into 48 well plates on the preconditioned BRL feeder cells.

Example 4: Primary and derivation culture of Houbara PGCs.

Single cell suspension of Houbara germinal crescent tissue were seeded into one well on a 48 well culture plate with mitotic inactivated BRL feeder cells monolayer, in the C0₂ incubator (37.0°C, 5%C0₂). The first media change was given on the third day. Subsequent media change interval was one third to half replacement every other day. Cultures were observed on day four and day seven of culture. PGCs were identified by morphological characteristics, e.g. rich granular inside cytoplasm, larger in size (15 - 28 pm). The number of PGCs was counted on day four, and the day prior to the first subculture, usually on day seven.

Houbara Bustard PGCs were not observed from the germinal crescent cell suspension (Figure 2 A). Typical morphological PGCs started appearing after three to four days in culture (Figure 2 B). The number of PGCs from male embryos on day four varied at a large range from 41 to 732, and on average 256.2+224.8 (n =1 7), while the number of PGCs from female embryos on day four varied from 1 to 1 193, on average 220.6±284.3 (n =20). A fewer number of PGCs were observed when the embryo was younger than stage 7. PGCs started proliferating once appeared; there was no morphological change during the first few divisions. Around day six to seven, the size of PGCs became smaller, and some PGCs formed small loose clumps of three to five cells sitting on top of the Houbara somatic cells (Figure 2 C, D). The rest of the PGCs existed in singular or doublet form.

Most of somatic cells from germinal crescent tissue attached on the feeder layers, and also started proliferating immediately after seeding. The somatic cells collected from the later stage (e.g., stage 9-10) proliferated very fast. It grew into 80% confluent after six to eight days culture. Example 5: Subsequent passages of Houbara Bustard PGCs culture: co-culture of PGCs with Houbara germinal crescent tissue derived somatic cells.

After five to seven days culture, the fibroblast like somatic cells derived from the Houbara germinal crescent grew very fast, and Houbara PGCs continued proliferating. Once they covered 80-90% of the feeders, the first passage was conducted.

1 . Half of the culture medium was replaced with the pre-warmed fresh medium.

2. To detach the PGCs from feeder layer and break up the small PGCs cluster or colonies, it was pipetted carefully with yellow tip.

3. The PGCs cells suspension was transferred into a 1 .5ml Eppendorf tube, and 1 50μΙ PBS (-) was added into the original well to rinse once.

4. 50μΙ 0.25% Trypsin- EDTA was added, and the cells were observed under inverted microscope until the stromal cells slightly detached and became round. An equal volume of culture medium was added to stop digestion.

5. All of the remaining cells were pipetted and harvested, and centrifuged to wash off remaining Trypsin-EDTA, and re-suspended the cells in PGCs culture medium.

6. A - 1/3 of the stromal cells were taken pooled into the PGCs suspension.

7. PGCs-somatic cells mixed suspension and the remaining stromal cells were seeded individually into in two different wells with feeder cells.

8. Upon observation on the 2^nd day, the well with stromal cells only was pipetted, and the unattached cells containing PGCs were transferred back to the other well with Houbara PGCs.

9. The PGCs and stromal cells were co-cultured on feeder cells for further four to five days until the stromal cells reached to confluent again. The next passages were conducted in the same process.

During the 7th-1 5th day, the majority of PGCs divided incompletely, and formed chain, shish kebab shape, or clumps tightly attached on the feeder layer. The next a few days, PGCs began to lose their typical morphology, e.g. the cytoplasmic granules disappeared, and cells divided one or two times into connected smaller cells with about half of their original size. The cells stopped proliferating after that stage (Figure 3 A, B, C). However, with the co-culture of Houbara PGCs and stromal cells on BRL feeder layer, a small portion PGCs kept their typical morphological characteristics and proliferated in single cell or clump form (Figure 3 D, E, F). These PGCs grew slowly, and divided nearly once a week.

The balance of Houbara Bustard somatic cells and BRL feeder cells was carefully maintained. After a month with four to five passages, the somatic cells grew slower, and finally diluted. Then, PGCs were passaged on the BRL feeder cells only (Figure 3 G, H). Male PGCs showed superior properties to female PGCs with a higher starting number of PGCs in primary culture, and more proportion of cells which divided into two single cells. There is no apparent behavior difference between male and female PGCs in the primary culture. However, almost all of the female Houbara Bustard PGCs stopped proliferating and lost their typical PGCs morphology during the second week of culture. The remaining cells formed tightly compacted clumps and attached on the feeder layer (Figure 3 C, D), and as such made it difficult to passage.

In this present example, a total number of 65 cultures were settled up (31 males and 34 females). 5 male Houbara Bustard PGC lines were established. The total cell number of each PGC cell line reached to over 1 -2 million after two to three months culture, and PGCs were maintained routinely and frozen down in liquid nitrogen.

Example 6: Maintenance culture of Houbara Bustard PGCs Established Houbara PGCs were routinely maintained in PGCs culture medium on mitotically inactivated BRL feeders. When confluent, cells can be dissociated by mechanically pipetting or enzymatically treated with Trypsin-EDTA, and passaged every five to seven days as following: The culture medium was removed, and the remaining medium was washed off with 100μΙ PBS(-). 50μΙ 0.25% Trypsin-EDTA was added, and incubated at room temperature for 30-50 seconds, and observed under the microscope, until PGCs inside the clump rounded up, and the border between cells was clearly visible. 1 0ΟμΙ culture medium was immediately added to stop digestion. The culture was gentle pipetted with a blue tip from the side of the well, to detach and break the PGCs clumps from the feeder layer. The cell suspension was harvested and centrifuged at 1450 rpm for five minutes at 4°C, and the pellet was re-suspended with fresh medium. Mechanically, the cultures were simply pipetted with the yellow tip, and observed under the microscope, until all the PGCs clumps were detached and broke into single cells or three to five cell small clumps. The cell suspension was harvested and centrifuged at 1450 rpm for five minutes at 4°C, and the pellet was re-suspended in fresh medium.

Cells were counted using a hemocytometer, and adjusted into a concentration of 3-5 x1 0⁵ cells/ml. 500 μΙ cell suspension per well were seeded in a 24 well plate. The cell number doubling time of Houbara PGCs in culture were calculated from five continuous passages. Cells were maintained or frozen down with freezing medium (growth media with 10% DMSO).

In maintenance culture, PGCs were seeded initially in uniform single cells. After overnight culture, PGCs migrated and formed four to five cell clusters with adjacent cells. During the rest of the culture period, the cells in the cluster compacted tightly in clump form. The majority of Houbara PGCs were in clump form, with some single form PGCs scattered among the clumps. Houbara PGCs were maintained over a year in this manner. The doubling time of Houbara PGCs in present cultures is 149.5+1 9.1 hours. There was no apparent morphology or proliferation kinetics change found (Figure 4 A, B). Example 7: Comparison of cultures of Houbara PGCs from germinal crescent tissue and migratory PGCs from circulating embryonic blood (stage 14)

To compare the effect of a different source of starting material on the Houbara PGCs derivation, PGCs from germinal crescent and migratory PGCs from embryonic circulation were cultured under the same conditions. Houbara germinal crescent tissue was collected and processed as described above. Ten freshly laid Houbara eggs were incubated under the above conditions for about 80 hours. 2- 5 μΙ embryonic blood containing migratory PGCs, was collected with a fine glass needle from dorsal aorta of Houbara embryos at stage 14-1 5 (H&H). Both germinal crescent cell suspension and blood samples were mixed into 300μΙ PGCs culture medium, and seeded on the preconditioned BRL feeder cells. Cultures were maintained for 20-30 days.

Blood source PGCs with typical PGCs morphology were observed (Figure 5A). Cells started to divide immediately after seeding. After four days culture, most blood PGCs were in doublet or two to three cells clumps, or three to five cells chain like structure (Figure 5 B). However, most of blood source PGCs started losing granules and stopped dividing from days five to seven (Figure 5 C, D). During the next two weeks culture, blood cells gradually died off. However, there were no typical PGCs observed any more from all the 10 cultures (Figure 5 E, F).

In contrast, in germinal crescent PGCs cultures, cells behaved as described in Example 5. Even though some PGCs lost their PGCs morphology, clumped, and attached, some typical PGCs could still be seen, divided in single cells form or in clump rich in granular in cytoplasm. These cells are promising to grow continuously. Thus, germinal crescent source PGCs showed better potential than migratory PGCs from embryonic circulation for derivation of a long-term culture and/or a PGCs line in vitro.

Example 8: Karyotype of long-term cultured Houbara Bustard PGCs To monitor the cytogenetic karyotype of Houbara PGCs after long-term culture, karyotyping of culture PGCs was conducted by preparing chromosome metaphase spreads. Briefly, Houbara PGCs were cultured for over eight months in the present system. Karyomax colcemid (Gibco 1 5210-040) was added at a final concentration of 0.1 pg/ml to PGCs culture in a 24 well plate, and incubated for one hour. After the incubation, the medium was removed, and the cells were washed with PBS(-). Houbara PGCs single cell suspension was harvested with Trypsin-EDTA disassociation. The cell suspension was spun down. Supernatant was removed, and the PGCs pellet was carefully mixed in the remaining solution. Hypotonic solution (0.56 % KCI) was added slowly (drop wise) to each sample and the tube was gently tapped to mix the contents. Cells were incubated in a 37°C waterbath for eight minutes, then centrifuged and gently re-suspended in the remaining supernatant. Fixative solution (75 % Methanol, 25% Acetic Acid) was added to each sample while mixing. Cells were incubated on ice for 20 minutes then washed twice with fixative by centrifugation. 20μΙ of the cells/fixative were dropped onto each slide. Once the slides dried, they were stained with Giemsa stain for 30 minutes. Spreads were finally mounted in DPX for observation. For G-Banding, slides with spreads were incubated at 90°C for 30 minutes, then dipped in PBS (-) at 37°C for two minutes, treated with 0.025% trypsin in PBS (-) at 37°C for 20-30 seconds, treated with 0.1 M Sorensen buffer pH 6.8, stained with 6 % Giemsa for four minutes, and mounted with DPX for observation.

28-50 spreads were counted and paired from each Houbara PGCs culture. The number of chromosomes of Houbara Bustard PGCs is about 62(2n). Though it is difficult to pair the small and micro-chromosome by shape, the spread of cultured Houbara Bustard PGCs showed diploid karyotype from the chromosomes which could be paired (Figure 6).

Example 9: Molecular analysis of the expression profile of pluripotency related and germ cells specific genes in cultured Houbara Bustard PGCs

PGCs are unipotent stem cells. Some stem cells related genes were found expressed in the in vivo and in vitro cultured chicken or mammalian PGCs. When being cultured in vitro, mammalian PGCs kept proliferating and transformed into embryonic germ cells, which were pluripotent and lost their germ cell identity. In the present example, Houbara Bustard PGCs were cultured over the long-term, and still kept the PGCs morphological characteristics. The expression of stem cells related and germ cells specific genes were analyzed by means of RT- PCR (Reverse transcriptase PCR), since the Houbara genomic sequence is not available yet. In this present example, the primers for detecting the expression of chicken cPOUV, cNANOG, cKLF4, cSOX2 genes were applied. Primers to detect the expression of Dazl, Myc, Vasa homolog genes were designed on the basis of the falcon gene sequence (http://www.ncbi.nlm.nih.gov/). Primer sequences are shown below in Table 4:

After more than eight months of culture, Houbara PGCs (HB61 ) were harvested. RNA isolation was performed using the RNeasy mini kit (Qiagen) with the aid of the DNase digestion kit to eliminate DNA contamination. All the process followed the instructions. Extracted RNA sample was stored at -80°C until further processing.

Reverse transcription of the cultured Houbara Bustard PGCs RNA was obtained using RT Ipsogen kit (Qiagen). ^g of RNA (10μΙ) was incubated at 65°C for 5 minutes. The sample was cooled immediately on ice for 5 minutes; then centrifuged briefly (10 seconds at 1000 rpm) and kept on ice. Reverse transcription premix was prepared as following: for a total volume of 1 5 μΙ, 5.0 μΙ reverse transcription buffer (5x), 2.0 μΙ dNTP 10 mM each, 5.25 μΙ random nanomer 100 μΜ, 0.5 μΙ RNase inhibitor 40 U/μΙ, 1 .0 μΙ reverse transcriptase 200 U/μΙ and 1 .25 μΙ DTT. 1 5 μΙ of the premix was added to the RNA sample. The sample was run on a thermal cycler using the following program:

reverse transcription 1 : 25°C,1 0 minutes;

reverse transcription 2: 50°C, 60 minutes;

Inactivation: 85°C, 5 minutes;

iv. cooling at 4°C, 5 minutes.

Finally, the sample was centrifuged briefly, then stored at-20°C until PCR.

PCR was performed following the Taq PCR master mix kits manual (Qiagen). A total of 25 μΙ reaction mix per sample was prepared: 12.5 μΙ master mix, 1 .25 μΙ primer forward, 1 .25 μΙ primer reverse, 8 μΙ H₂0, 2 μΙ Houbara Bustard PGCs cDNA. The reaction complete mix was then quickly pipetted to mix, centrifuged and placed in the thermal cycler. PCR (with 40 cycles) was run at 95°C for 20 minutes, 95°C for 30 seconds, 55 - 59°C for 30 seconds (annealing temperature 55°C for amplification of Dazl, Myc, Vasa, cPOUV, cNANOG, cKLF4, and cSOX2), 72°C for 1 minute, and 60°C for 30 minutes. PCR products were loaded on 1 % agarose gel in TBE buffer and run for 40 minutes at 60 V. To confirm the specificity of amplification, PCR product was sequenced and compared with the homologue genes of other species using BLAST.

Present results showed that pluripotent related genes (Pouv, Nanog, Klf4, Sox2,Myc) were expressed in long-term cultured Houbara PGCs and germ cells related homolog genes (Dazl and Vasa) were also expressed (Figure 7).

Example 10: Immunofluorescent staining of Houbara PGCs with anti-CVH

VASA is an RNA binding protein with an RNA dependent helicase. The vasa gene is essential for germ cell development and was first identified in Drosophila melanogaster. It is homologous to a DEAD (Asp-Glu-Ala-Asp)-family protein in the mouse. In early embryogenesis, vasa is involved in abdomen development, nanos mRNA translation, and pole plasm and germ cell formation. In oogenesis, vasa is involved in germline cyst development, oocyte differentiation, gurken mRNA translation, oocyte polarity and translation of oskar mRNA. Chicken vasa homolog (CVH) gene was identified in 2000 (Tsunekawa N, Naito M, Sakai Y, Nishida T and Noce T. Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells. Development, 127: 2741 -2750. 2000), and expressed in PGCs and adult germ cells. Anti-CVH

Houbara PGCs were cultured, and single cell suspension was harvested. Chicken PGCs were cultured under conditions as published (Van de Lavoir MC, Diamond JH, Leighton PA, Mather-Love C,Heyer BS, Bradshaw R, Kerch ner A, Hooi LT, Gessaro TM, SwanbergSE, Delany ME and Etches RJ. Germline transmission of genetically modified primordial germ cells. Nature, 441 : 766-769. 2006), which is incorporated by reference herein, and harvested as positive control. Cells were fixed in 4 % PFA for 15 minutes. Smears were prepared on TESPA coated slides. Cells were permeabilized with 0.5 % triton x-100 for 10 minutes at room temperature, and incubated for 45-60 minutes in blocking buffer (PBS/5% BSA/0.5% tween- 20). Primary antibody Rabbit a CVH (1 :500) was added and incubated overnight in a humidified chamber at 4°C (Tsunekawa N, Naito M, Sakai Y, Nishida T and Noce T. Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells. Development, 127: 2741 -2750. 2000). Cells were washed with PBS (-), incubated with Goat cxRabbit biotinylated (1 :200) for 60 minutes at room temperature. Texas Red® Avidin D (fluorescent Avidin kit A-1 100, vector laboratories) was added for 60 minutes at room temperature, then washed with PBS (-), finally mounted with Vectashield mounting media with DAPI.

The results showed that both, chicken PGCs and Houbara PGCs, were also stained positively (Figure 8). It suggested that Houbara PGCs still kept their germ cells identity after over a year's culture.

Example 1 1 : Transfection of cultured Houbara PGCs with Piggybac transposon system carrying GFP gene. To visualize the Houbara PGCs in vivo after transplantation, exogenous GFP gene was introduced into cultured Houbara PGCs by Piggybac transposon system (Macdonald J, Taylor L, Sherman A, Kawakami K, Takahashi Y, Sang HM and McGrew MJ. Efficient genetic modification and germ-line transmission of primordial germ cells using piggy-Bac and Tol2 transposons. Proceedings of the National Academyof Sciences of the USA, 109: 1466-1472. 2012). Cells were transfected by electroporation using pipette-type electroporator (Invitrogen). Briefly, a total of 3x10⁴ PGCs was re-supended in 9 μΙ Neon electroporation buffer (Invitrogen), and 0.2 μg of plasmid Piggybac transposon system was added. The wave pulse was set as 850 v, 40 ms, one pulse (Establishment of an efficient and stable transgene expression system in chicken primordial germ cells. Ju Hyun Yang and Sungtae Kim, Bull. Korean Chemi.Soc.2012, 33 (5): 1536-1 540). The transient transfection rate was evaluated by counting the green fluorescent cells number on the second day of transfection. After transfection, cells were continued culturing for two weeks before selection. Selection was performed for two weeks by adding puromycin to culture medium at final concentration of 0.75 g/ml. Houbara PGCs were successfully transfected (Figure 9). The transient transfection rate was approximately 5.1 %. Example 12: Transferring long-term cultured Houbara PGCs into chicken embryo at stage X and stage 15-16

Houbara PGCs were cultured as described above. Media were removed carefully and PGCs, which slightly attached to the feeder layer, were rinsed with PBS (-). Cells were harvested after dissociated with Trypsin EDTA. PGCs single cell suspension was adjusted into a concentration of 5x1 OYrnl with growth media, and kept at room temperature.

Injection of Houbara Bustard PGCs into blastoderm of chicken embryo (stage X)

Freshly laid White Leghorn chicken eggs were used as the recipient. A window with a diameter of 1 cm was made by removing piece of eggshell from the blunt end. The inner shell membrane was removed to expose the blastoderm. 5000 Houbara PGCs in 1 μΙ of culture medium were injected into the subgerminal cavity. Windowed eggs were fully filled with albumin, sealed with cling wrap and tightly covered with a cell culture dish. Eggs were put back into the incubator with the sharp end up. After 20 hours of incubation, the eggs were opened. The whole embryo and peripheral tissue was dissected, and observed under fluorescence microscope to check the distribution of Houbara PGCs. The remaining eggs were opened on day three. 2ml eggwhite was carefully removed, and resealed with cling wrap. Some embryos were sacrificed on day eight, the gonadal tissue was dissected, and Houbara cells were observed and counted under fluorescence microscope. The remaining embryos continued incubating until feather fully developed beyond 14 days. A total of 74 eggs were injected. Five embryos were sacrificed and observed under fluorescence microscope after 24hours incubation. Houbara PGCs were found from 80.0 % (4/5) of the embryos. All of the Houbara Bustard PGCs were located in the germinal crescent area of the chicken embryo (Figure 10 A), i.e. Houbara PGCs were not found otherwise in the body of chicken embryo (stage 8-10). Gonadal tissues were dissected from 1 6 injected chicken embryos on day eight, and digested into singe cell suspension. 93.4% (1 5/1 6) of chimeric chicken embryo showed Houbara PGCs in their gonad; and the number of Houbara PGCs ranged from 24 to 1026, with the average of 290.5+31 8.5; and 200.4+243.3 in the male embryos (n= 9), 406.3±383.2 in the female embryos (n= 7). Many references have reported that some chicken PGCs in culture spontaneously transformed into pluripotent embryonic germ cells, which confirmed to form phenotypic chimera. In present culture, Houbara PGCs clumped together, and some of clumps attached on the feeder cells, though no morphological EG cells colonies were observed. 25 embryos developed beyond 14 days with fully feather development, no phenotypic chimera were observed. Injection of Houbara Bustard PGCs into blood circulation of chicken embryo (stage 15-16) Fertilized White Leghorn chicken embryos were incubated to stage 1 5-1 6 for 58 hours at 37.8°C, 60 % relative humidity. To expose the embryo, a small window was opened by removing pieces of eggshell from the sharp end. A number of 5000 Houbara PGCs was injected into the dorsal aorta of chicken embryos with a fine glass needle. Injected eggs were sealed with double layer of parafilm. Eggs were returned to the incubator, and continued incubation for 5 days till stage 33. Gonadal tissue were dissected and observed under inverted fluorescence microscope, then mechanically and enzymatically dissociated into single cell suspension. The number of green Houbara PGCs was counted. A total number of 30 eggs were injected. 23 embryos survived on day 8. Gonadal tissues were dissected from 15 embryos. All the 1 5 chicken embryos showed Houbara PGCs in the gonads; and the number of Houbara PGCs ranged from 658 to 3475, with the average of 1 844.1 +751 .1 ; and 21 67.9±808.3 in the male embryos (n= 7), 1 560.8+609.7 in the female embryos (n= 8). The majority of green Houbara PGCs were located in the left testis of chimeric chicken embryo (Figure 10 B, C). Example 13: Germline transmission of long-term cultured Houbara PGCs.

Freshly laid Houbara eggs were incubated for 80-85 hours with the blunt end up at 37.8°C in a dry incubator. Eggs were opened from the sharp end to expose embryos. A number of 5x10³ Houbara green PGCs (HB513g, male) with a total culture period of over 1 0 months) in 1 μΙ growth media were injected into the recipient Houbara embryos (stage 15-1 6, H&H). Injected eggs were sealed with double layers of parafilm, and returned back to the incubator to hatch. Eggs were candled daily after 14 days incubation. Gonadal tissue were dissected from the dead embryos, and observed under fluorescence microscope. A total number of 1 8 Houbara embryos were injected, 55.6% (10/18) embryos survived beyond three weeks, and 1 1 .1 % (2/1 8) of the injected embryo hatched. One chick died a week after hatching. Houbara Bustard PGCs derived germ cells were found from five gonadal samples (male in Figure 1 1 , female in Figure 12). In the testis, most donor green cells were found in the left gonad, and in the ovary tissue, cells were found in the cortex area. The present results suggested that Houbara Bustard PGCs could still keep the capability to migrate and proliferate in recipient gonad, even after long-term in vitro culture.

Claims

1 . Method for establishing a long-term culture of avian primordial germ cells, characterized in that the primordial germ cells are derived from germinal crescent tissue isolated from an avian embryo at stage 4 to 1 1 , preferably at stage 4 to 1 0, more preferably at stage 6 to 10, even more preferably at stage 7 to 10.

2. Method for establishing a long-term culture of avian primordial germ cells according to claim 1 , characterized in that the avian embryo is a male avian embryo.

3. Method for establishing a long-term culture of avian primordial germ cells according to claim 1 or 2, characterized in that the germinal crescent tissue is dissociated after its isolation from the embryo to obtain a cell suspension of germinal crescent tissue.

4. Method for establishing a long-term culture of avian primordial germ cells according to any of claims 1 to 3, comprising the following steps:

a) Isolating germinal crescent tissue from an avian embryo at stage 4 to 1 1 , preferably at stage 4 to 10, more preferably at stage 6 to 10, even more preferabl at stage 7 to 10;

b) Establishing of a primary culture by culturing cells of the germinal crescent tissue on a feeder matrix;

d) transferring the primordial germ cells to a culture largely devoid of, preferably without, somatic cells derived from germinal crescent tissue.

5. Method for establishing a long-term culture of avian primordial germ cells according to claim 4, characterized in that the method comprises step c).

Method for establishing a long-term culture of avian primordial germ cells according to claim 4 or 5, characterized in that the feeder cells are mouse fibroblasts, preferably STO cells, and/or buffalo rat liver cells, whereby buffalo rat liver cells are preferred.

Method for establishing a long-term culture of avian primordial germ cells according to any of claims 4 to 6, characterized in that a transfer of the cells following step b) comprises the sub-steps of

(i) harvesting primordial germ cells; and

Method for establishing a long-term culture of avian primordial germ cells according to claim 7, characterized in that after sub-step (ii) a mixture of the primordial germ cells and the somatic cells derived from germinal crescent tissue is prepared, whereby the mixture preferably comprises 10 % to 50 %, more preferably 20 % to 40 %, and even more preferably 25 % to 33 %, of the cells harvested in sub-step (ii), and the mixture is seeded in a vessel containing a feeder matrix.

9. Method for establishing a long-term culture of avian primordial germ cells according to claim 8, characterized in that the mixture is prepared and seeded in each of the transfers of the cells in step c).

10. Method for establishing a long-term culture of avian primordial germ cells according to any of claims 1 to 9, characterized in that the avian primordial germ cells are primordial germ cells from bustard, chicken, duck, quail, hawk, or ibis, e.g. northern bald ibis (waldrapp), preferably from bustard, in particular from houbara bustard and/or that the avian primordial germ cells are primordial germ cells from an endangered bird.

1 1 . An isolated avian primordial germ cell, characterized in that the primordial germ cell is derived from germinal crescent tissue isolated from an avian embryo at stage 4 to 1 1 , preferably at stage 4 to 10, more preferably at stage 6 to 1 0, even more preferably at stage 7 to 10.

12. Culture of an avian primordial germ cell according to claim 1 1 .

1 3. Culture of an avian primordial germ cell according to claim 12, characterized in that in that the culture further comprises a feeder matrix and a culture medium, the culture medium comprising the growth factors FGF₂, SCF, LIF, and IGF-1 and preferably comprising a conditioned medium, more preferably feeder cell conditioned medium, even more preferably BRL conditioned medium.

14. Culture of an avian primordial germ cell according to claim 1 3, characterized in that in that the culture medium further comprises a basal medium, preferably DMEM, more preferably knockout DMEM, animal serum, preferably FBS and/or chicken serum, an antibiotic, preferably penicillin/streptomycin, and further additives, preferably including nucleosides, a glutamine derivative, preferably GlutaMax, MEM- NEAA, a biological buffer, preferably HEPES, pyruvate (e.g. sodium pyruvate), and/or β-mercapto-ethanol .

1 5. Culture of an avian primordial germ cell according to any of claims 12 to 14, characterized in that the avian primordial germ cells are cultured in vitro for at least 5 days, at least 10 days, at least 14 days, at least 25 days, at least 50 days at least 75 days, at least 100 days, at least 1 50 days, at least 200 days, at least 250 days, at least 300 days, and/or at least 350 days.

1 6. Cell line derived from an avian primordial germ cell according to claim 1 1 .

1 7. Cell line derived from an avian primordial germ cell according to claim 1 6, which is a cell line of primordial germ cells of a male bird.

18. Cell line derived from an avian primordial germ cell according to claim 1 6 or 1 7, characterized in that the total cell number of the cell line after three months in vitro culture is more than 1 million cells, preferably more than 1 ,5 million cells, more preferably more than 2 million cells, even more preferably more than 2,5 million cells, and particularly preferably more than 3 million cells.

19. An isolated avian primordial germ cell according to claim 1 1 , a culture of an avian primordial germ cell according to any of claims 12 to 15, or a cell line derived from an avian primordial germ cell according to any of claims 1 6 to 1 8, characterized in that the primordial germ cells are bustard primordial germ cells, preferably houbara bustard primordial germ cells.

20. An isolated avian primordial germ cell according to claim 1 1 or 19, a culture of an avian primordial germ cell according to any of claims 12 to 1 5 and 1 9, or a cell line derived from an avian primordial germ cell according to any of claims 1 6 to 1 9, characterized in that the avian primordial germ cell is genetically modified.

21 . An isolated avian primordial germ cell, a culture of an avian primordial germ cell or a cell line derived from an avian primordial germ cell according to claim 20, characterized in that the genetic modification is the introduction of a reporter gene and/or of a disease resistance into an avian primordial germ cell.

22. Method for genetically modifying an isolated avian primordial germ cell according to claim 1 1 or any of claims 1 9 to 21 , a culture of an avian primordial germ cell according to any of claims 12 to 1 5 or any of claims 1 9 to 21 , or a cell line derived from an avian primordial germ cell according to any of claims 1 6 to 21 , characterized in that a transposon system, preferably a PiggyBac Transposon system, is used.

23. Use of an isolated avian primordial germ cell according to claim 1 1 or any of claims 19 to 21 , a culture of an avian primordial germ cell according to any of claims 12 to 1 5 or any of claims 19 to 21 , or a cell line derived from an avian primordial germ cell according to any of claims 16 to 21 for genetic modification of the avian primordial germ cell.

24. Use of an isolated avian primordial germ cell, a culture of an avian primordial germ cell or a cell line derived from an avian primordial germ cell according to claim 23, characterized in that the genetic modification is the introduction of a reporter gene and/or a disease resistance into the avian primordial germ cell.

25. Use of an isolated avian primordial germ cell according to claim 1 1 or any of claims 19 to 21 , a culture of an avian primordial germ cell according to any of claims 12 to 15 or any of claims 19 to 21 , or a cell line derived from an avian primordial germ cell according to any of claims 16 to 21 for obtaining a germline chimera.

26. Use of an isolated avian primordial germ cell according to claim 1 1 or any of claims 1 9 to 21 , a culture of an avian primordial germ cell according to any of claims 12 to 1 5 or any of claims 19 to 21 , or a cell line derived from an avian primordial germ cell according to any of claims 1 6 to 21 for preservation and/or mass production of the bird, the bird being preferably of an endangered avian species.

27. Use of a culture of an avian primordial germ cell according to any of claims 12 to 1 5 or any of claims 1 9 to 21 , or a cell line derived from an avian primordial germ cell according to any of claims 1 6 to 21 for preserving the (genetic) diversity of an endangered avian species, comprising a method for establishing a long-term culture of avian primordial germ cells according to any of claims 1 to 10.

28. Use of a culture of an avian primordial germ cell or a cell line derived from an avian primordial germ cell according to claim 27 further comprising a step of cryopreservation.

29. Culture medium for culturing avian primordial germ cells, preferably for culturing houbara bustard primordial germ cells, comprising a basal medium, preferably DMEM, more preferably knockout DMEM; a conditioned medium, preferably feeder cell conditioned medium, more preferably BRL conditioned medium; animal serum, preferably FBS and/or chicken serum; the growth factors FGF₂, SCF, LIF, and IGF-1 ; preferably an antibiotic, more preferably penicillin/streptomycin; and preferably further additives, including for example nucleosides, a glutamine derivative, preferably GlutaMax, MEM-NEAA, a biological buffer, preferably HEPES, pyruvate (e.g. sodium pyruvate), and/or β-mercapto-ethanol.

30. Method for maintaining an isolated avian primordial germ cell according to claim 1 1 or any of claims 1 9 to 21 , a culture of an avian primordial germ cell according to any of claims 12 to 1 5 or any of claims 1 9 to 21 , or a cell line derived from an avian primordial germ cell according to any of claims 16 to 21 , characterized in that the avian primordial germ cells are maintained in a culture medium on a feeder matrix, the culture medium comprising the growth factors FGF₂, SCF, L1F, and IGF-1 and preferably a conditioned medium, more preferably feeder cell conditioned medium, even more preferably BRL conditioned medium.

31 . Method for maintaining an isolated avian primordial germ cell, a culture of an avian primordial germ cell, or a cell line derived from an avian primordial germ cell according to claim 30, characterized in that the avian primordial germ cells are maintained in a culture medium according to claim 29.

32. Method for maintaining an isolated avian primordial germ cell, a culture of an avian primordial germ cell, or a cell line derived from an avian primordial germ cell according to claim 31 , characterized in that the culture medium further comprises 10% DMSO and the method further comprises a step of freezing a culture of avian primordial germ cells.

33. Method for molecular analysis of an avian primordial germ cell, in particular for molecular analysis of a Houbara bustard primordial germ cell, characterized in that an oligonucleotide is used, in particular as a primer and/or a probe, said oligonucleotide comprising or consisting of a sequence according to any of SEQ ID NO: 1 to SEQ ID NO: 6 or sequence variants thereof.

34. Method for molecular analysis of an avian primordial germ cell according to claim 33, characterized in that the method comprises a step, wherein a nucleic acid of the avian primordial germ cell is amplified using a primer pair selected from the group consisting of:

1 ) an oligonucleotide comprising or consisting of a sequence according to SEQ ID NO: 1 or sequence variants thereof and an oligonucleotide comprising or consisting of a sequence according to SEQ ID NO: 2 or sequence variants thereof;

2) an oligonucleotide comprising or consisting of a sequence according to SEQ ID NO: 3 or sequence variants thereof and an oligonucleotide comprising or consisting of a sequence according to SEQ ID NO: 4 or sequence variants thereof; and

3) an oligonucleotide comprising or consisting of a sequence according to SEQ ID NO: 5 or sequence variants thereof and an oligonucleotide comprising or consisting of a sequence according to SEQ ID NO: 6 or sequence variants thereof.

An oligonucleotide comprising or consisting of a sequence selected from the group consisting of: a sequence according to SEQ ID NO: 1 or sequence variants thereof, a sequence according to SEQ ID NO: 2 or sequence variants thereof, a sequence according to SEQ ID NO: 3 or sequence variants thereof, a sequence according to SEQ ID NO: 4 or sequence variants thereof, a sequence according to SEQ ID NO: 5 or sequence variants thereof and a sequence according to SEQ ID NO: 6 or sequence variants thereof.

Primer pair for molecular analysis of a nucleic acid of an avian primordial germ cell comprising at least one oligonucleotide according to claim 35.

Method for obtaining an interspecies or intraspecies germline chimera comprising the following steps:

(i) Establishing a long-term culture of avian primordial germ cells according to any of claims 1 to 10 or maintaining an isolated avian primordial germ cell, a culture of an avian primordial germ cell, or a cell line derived from an avian primordial germ cell according to to any of claims 30 to 32; and (ii) Providing an interspecies or intraspecies germline chimera by introducing the primordial germ cell of step (i) into a recipient of the same or different avian species.

38. Method for obtaining an interspecies or intraspecies germline chimera according to claim 37, characterized in that the cultured primordial germ cells of step (i) are cultured in vitro ior at least 5 days, at least 10 days, at least 14 days, at least 25 days, at least 50 days at least 75 days, at least 1 00 days, at least 1 50 days, at least 200 days, at least 250 days, at least 300 days, and/or at least 350 daysand/or even longer, before they are introduced into a recipient in step (ii).

39. Method for obtaining an interspecies or intraspecies germline chimera according to claim 37 or 38, characterized in that the cultured primordial germ cells of step (i) are genetically modified.

40. Method for obtaining an interspecies or intraspecies germline chimera according to any of claims 37 to 39, characterized in that the donor of the avian primordial germ cells is an endangered bird, and/or the donor of the avian primordial germ cells is of the order Cruiformes, preferably of the family Otididae (Bustards), more preferably of the genus Chlamydotis, and even more preferably the donor of the avian primordial germ cells is a houbara bustard {Chlamydotis undulata).

41 . Method for obtaining an interspecies or intraspecies germline chimera according to any of claims 37 to 40, characterized in that the recipient is a chicken, preferably a chicken of White Leghorn strain, or a bird of the order Gruiformes, preferably of the family Otididae (Bustards), more preferably of the genus Chlamydotis and even more preferably a houbara bustard Chlamydotis undulata).

42. Germline chimera having a germ line comprising a primordial germ cell derived from a culture of an avian primordial germ cell according to any of claims 12 to 15 or any of claims 19 to 21 , or from a cell line derived from an avian primordial germ cell according to any of claims 1 6 to 21 .

43. Germline chimera according to claim 42, characterized in that the primordial germ cell derived from the culture of an avian primordial germ cell according to any of claims 12 to 1 5 or any of claims 1 9 to 21 , or from the cell line derived from an avian primordial germ cell according to any of claims 1 6 to 21 are primordial germ cells derived from an endangered bird, and/or from a bird of the order Cruiformes, preferably of the family Otididae (Bustards), more preferably of the genus Chlamydotis, and even more preferably the primordial germ cell is derived from a houbara bustard {Chlamydotis undulata).

44. Method for obtaining a pure species offspring from an interspecies germline chimera comprising the following steps:

(ii) Cross-breeding the interspecies germline chimera with

b. an interspecies germline chimera obtained by a method according to any of claims 37 to 41 , wherein the species of the donor of the introduced avian primordial germ cells and the species of the recipient embryo correspond to the species of the donor of the introduced avian primordial germ cells and the species of the recipient embryo of the interspecies germline chimera obtained in step (i); and (iii) selecting pure species offspring, which is preferably of the same avian species as the donor of the primordial germ cells introduced in the interspecies germline chimera in step (i).

45. Method for obtaining a pure species offspring from an interspecies germline chimera according to claim 44, characterized in that the pure species offspring is an endangered bird and/or a bird of the order Cruiformes, preferably of the family Otididae (Bustards), more preferably of the genus Chlamydotis and even more preferably a houbara bustard {Chlamydotis undulata).