JP2003525581A - Vertebrate testis cell depopulating method and method for producing transgenic species - Google Patents

Abstract

  (57) [Summary] Compositions for in vitro and in vivo transferrin of vertebrate male germ cells are inter-analyzed by nucleic acids or transgenes, and gene transfer systems, and optionally with endosomal lysates, transgenes, and the nucleus of male germ cells A protective inter-analyzing substance, such as a virus or viral component that enhances gene transfer through the cytoplasm into the cell. Methods for genetically modifying vertebrate male germ cells in vivo utilize lentivirus-derived vectors. Methods for substantially reducing vertebrate testes use a combination of gamma irradiation doses with doses of alkylating agents such as busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylethanesulfonic acid. Pharmaceutical preparations and transfer kits make use of the present compositions. A method of introducing a polynucleotide into a vertebrate male germ cell comprises administering the composition to a vertebrate. Methods for isolating or selecting transfected cells utilize the reporter gene, and methods for administering transfected male germ cells utilize in vitro transfected male germ cells.

Description

Detailed Description of the Invention

BACKGROUND OF THE INVENTION Throughout this application, various publications are referenced in parentheses. The disclosures of these publications are incorporated herein by reference in their entirety to more fully describe the state of the art to which this invention belongs.

1. FIELD OF THE INVENTION The present invention relates to the field of medicine, in particular transgenic animals and gene therapy. The present invention is particularly directed to in vitro and in vivo methods of transfecting male germ cells and supporting cells (ie Leydig cells and Sertoli cells), which methods include methods of depleting male germ cells from vertebrate testes.

2. Discussion of Related Fields The field of transgenic animals was initially developed to understand the actions of single genes and the phenomena of gene activation, expression and interaction in whole animals. This technique has been used to model various diseases in humans and other animals. Transgenic technology is one of the most powerful tools available for genetic research as well as for understanding genetic mechanisms and functions. The technique is also used to investigate the relationship between genes and diseases. About 5,000
The disease is caused by a single genetic defect. More generally, other diseases are the result of complex interactions of one or more genes with environmental agents such as viruses or carcinogens. Understanding such interactions is of paramount importance for the development of therapeutics such as gene therapy and drug treatment, and treatments such as organ transplantation. Such treatments may compensate for functional deficits and / or eliminate unwanted function expressed in the organ. Gene transfer has also been used for livestock improvement and large-scale production of bioactive agents.

Historically, transgenic animals have been produced almost exclusively by microinjection of fertilized eggs. The pronucleus of a fertilized egg is microinjected with a foreign substance, that is, a heterologous or allogenic DNA or hybrid DNA molecule in vitro. Next, the microinjected fertilized egg is transferred into the genitals of the foster female animal. (For example, PJAKrimpenfort et al., “Transgenic Mice Depleted of Mature T Cells and Method of Making Transgenic Mice (
Transgenic mice depleted in mature T-cells and methods for making transg
enic mice), US Pat. Nos. 5,175,384 and 5,434,340; PJAKrimpenfort et al., “Transgenic mice depleted in mature lymphocytic cell-ty.
pe.) ", U.S. Pat. No. 5,591,669).

The production of transgenic animals according to the present technology is generally reproducible and for this reason little improvement has been made. However, this technique requires large numbers of fertilized eggs. Part of the reason for this is the high rate of fertilized egg loss due to lysis during microinjection. Moreover, engineered embryos are less likely to colonize the uterus and survive. Due to these factors, the efficiency of this technology is extremely low. For example, to produce 3 transgenic animals, perhaps 300 to 5 fertilized eggs
You may need to microinject to 00. Because of the need for microinjection into large numbers of embryos as part of that reason, transgenic technology is predominantly used in mice because of its high fertility. Small animals such as mice have proven to be suitable models for certain diseases, but their value in this regard is limited. Larger animals are much more suitable for studying the action and treatment of most human diseases because they are similar to humans in many respects and in organ size. Currently, transgenic animals with the potential for human xenotransplantation are under development, but larger animals of comparable size to humans will be needed. Transgenic technology will render such donor animals immunocompatible with human recipients. However, conventional transgenic techniques require large numbers of fertilized female germ cells or eggs. Most large mammals, such as primates, cows, horses and pigs, produce only a few fertilized eggs per animal cycle, even after hormone stimulation.
Only produce 10 to 20 pieces. As a result, the cost of producing large animals by these techniques is prohibitive.

The present invention is based on the fact that large numbers of male germ cells are more readily available. Most male mammals generally produce at least 10 ⁸ sperm (male germ cells) per ejaculation. This is in contrast to only 10-20 eggs in mice even after treatment with ovulation inducers. A similar situation holds true for ovulation in almost all large animals. Considering only this reason, male germ cells are
Introducing A into the germ line is a better target for transgenic animals with increased efficiency after simple natural mating.

[0007] Spermatogenesis is a self-propelled haploid cell that allows diploid spermatogonial stem cells to undergo dramatic and distinct morphological changes to fertilize an egg when fully mature (male The process of providing daughter cells to be gametes.

Primordial germ cells were initially found in the endodermal yolk sac epithelium at the E8 stage and are thought to arise from the ectoderm (A. McLaren and Buehr).
), Cell Diff. Dev. 31: 185 [1992]; Y. Matsui et al., Nature 353: 75.
0 [1991]). They move from the yolk sac epithelium through the hindgut endoderm to the genital ridge,
Proliferate by mitosis and settle in the testis.

Upon reaching sexual maturation, spermatogonia undergo meiosis 5 or 6 times before entering meiosis. Primordial spermatogonial stem cells (Ao / As) proliferate and grow into intermediate spermatogonia cell types Apr, Aa
l, forming a population of A1-4, after which they differentiate into type B spermatogonia. Type B spermatogonia differentiate to form primordial spermatocytes, which enter long meiotic prophase, during which homologous chromosomes pair and recombine. The morphologically distinguishable meiotic phases are: prefilament phase, filament phase, zygote phase, thick thread phase, secondary spermatocytes and haploid spermatids. Sperm cells undergo large morphological changes during spermatogenesis, such as nuclear remodeling, acrosome formation, and tail assembly (AR Bellve et al., Sperm Recovery, Capacitation, Acrosome reaction and fractionation (Recovery, capacitation, acrosome
reaction, and fractionation of sperm), Methods Enzymol. 225: 113-36.
[1993]). Spermatocytes and sperm cells establish life-sustaining contacts with Sertoli cells through their unique, semi-zygotic adhesion with the Sertoli cell membrane. The final changes in sperm maturation occur in the genitals of female animals before fertilization.

Initially, an attempt was made to add a DNA to sperm and use it to fertilize a mouse egg in vitro to produce a transgenic animal. Fertilized eggs were then transferred to foster females to give birth to 30% transgenics, which were reported to express the transgene. However, despite repeated efforts by other researchers, this experiment was not reproducible and no transgenic pups were obtained. In fact, it is almost certain that the transgenic animals that were suspected to have been obtained by this method were not transgenic at all and the reported DNA integration was merely an experimental artifact. Data collected from laboratories worldwide involved in testing this method showed that no transgenic animals were obtained from all 890 offspring.

In summary, although it is now possible to produce surviving transgenic offspring, the methods available are expensive and extremely inefficient. Sperm transfection according to the present invention, whether in vitro or in vivo,
It provides a simpler, cheaper, less invasive method for producing transgenic animals, which is extremely useful not only for transferring heterologous genes into the germ cells of animals, but also for transferring allogeneic genes. Provide a potentially efficient method.

In order to facilitate in vitro transfection of male germ cells and transplantation into the testes of recipient mouse male vertebrates, untransfected male germ cells are transferred prior to transfer of the transfected male germ cells therein. It is convenient to first deplete the germ cells from the recipient vertebrate testis.

Cytocytopenia of the testes is generally performed by exposing the whole vertebrate to gamma irradiation (X-rays) or by local irradiation of the testes. (For example, Pinon-
G. Pinon-Lataillade et al., Endocrinological and histological changes induced by continuous low-dose gamma irradiation of rat testis.
istological changes induced by continuous low dose gamma-irradiation of
rat testis) ", Acta Endocrinol. (Copenh) 109 (4): 558-62 [1985]; Pinone-
G. Pinon-Lataillade and J. Maas et al., "Continuous gamma irradiation in rats: dose-rate effect on spermatogenesis loss and recovery (Continuou
s gamma-irradiation of rats: dose-rate effect on loss and recovery of sp
ermatogenesis) ”, Stahlentherapie 161 (7): 421-26 [1985]; Hopkinson (
CR Hopkinson) et al., The effect of local testicular irradiati on testicular tissue and plasma hormone levels in male rats.
on on testicular histology and plasma hormone levels in the male rat.)
Acta Endocrinol. (Copenh) 87 (2): 413-23 [1978]; G. Pinon-Lataillade et al. Influence of germ cells upon Serto
li cells during continuous low-dose rate gamma-irradiation of adult rats
) ", Mol. Cell. Endocrinol. 58 (1): 51-63 [1988]; Kamchawing (P. K.
amtchouing) et al., "Effect of continuous low-dose rate gamma-irradiation on rat Se function.
rtoli cell function.) ", Reprod. Nutr. Dev. 28 (4B): 1009-17 [1988]; C. Pineau et al.," Evaluation of testis function after acute and chronic irradiation: Sertoli in adult rats. Further evidence for the effects of late sperm on cell function (
Assessment of testicular function after acute and chronic irradiation: f
urther evidence for influence of late spermatids on Sertoli cell functio
n in the adult rat), Endocrinol. 124 (6): 2720-28 [1989]; M. Kangasniemi et al. Cellular regulation of basal and FSH-stimul
ated cyclic AMP production in irradiated rat testes.) ", Anat. Rec. 227
(1): 32-36 [1990]; G. Pinon-Lataillade et al., "Effect of an acute exposure of rat on testicular and Sertoli and Leydig cell functions. testes to gamma rays
on germ cells and on Sertoli and Leydig cell functions.) '', Reprod Nut
r. Dev. 31 (6): 617-29 [1991]).

The mechanism of gamma-irradiation-induced spermatogonia degeneration is thought to be related to the process of apoptosis (M. Hasegawa et al., “Differentiated spermatogonia resistance to radiation-induced apoptosis and p53-deficient mice. In (Resis
tance of differentiating spermatogonia to radiation-induced apoptosis an
d loss in p53-deficient mice.) ", Radiat. Res. 149: 263-70 [1998]).

Another method of depleting vertebrate testis cells is by administering a composition comprising an alkylating agent such as busulfan (Milleran). (For example, FX Jiang, “Behaviour of spermatogonia following recovery from bus from recovery from busulfan treatment in rats.
ulfan treatment in the rat) ", Anat. Embryol. 198 (1): 53-61 [1998]; LD Russell and RL Brinster," Spermatogenesis after transplantation of rat testis cells into the mouse vas deferens. Ultrastructural knowledge about
observations of spermatogenesis following transplantation of rat testis
cells into mouse seminiferous tubules), J. Androl. 17 (6): 615-27 [199
6]; N. Boujrad et al., "Evolution of somatic and germ cell populations after busulfan tr in rat uterus or neonatal cryptochidism.
eatment in utero or neonatal cryptochidism in the rat.) ", Andrologia 2
7 (4): 223-28 [1995]; RE Linder et al., "Endopoint of sperm toxicity in rats after short-term exposure to 14 reproductive toxins.
permatotoxicity in the rat after short duration exposures to fourteen re
productive toxicants) ”Reprod. Toxicol. 6 (6): 491 ~ 505 [1992]: Kasuga &
F. Kasuga ans M. Takahashi, “The endocrine function of rat gonads with reduced number of germ cells after treatment with busulfan
with reduced number of germ cells following busulfan treatment.) ", En
docrinol. Jpn. 33 (1): 105-15 [1986]).

Cytotoxic alkylating agents such as busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylethanesulfonic acid are often used in cancer chemotherapy to kill malignant cells. (For example, Ande
rsson) et al., "Parenteral busulfane (Parenteral busulfane for the treatment of malignant diseases.
an for treatment of malignant disease) ", U.S. Patent Nos. 5,559,148 and
5,430,057; Stratford et al., "Stimulation of stem cell growth by the bryostatins.",
US Pat. No. 5,358,711; Luck et al., “Treatment employing vasoconstrictive with vasoconstrictors in combination with cytotoxic agents.
substances in combination with cytotoxic agents for introduction into c
ellular lesion.) ”, U.S. Pat. No. 4,978,332). Treatment of mice with busulfan (13-40 mg / kg body weight) has been reported to deplete male germ cells in the testis; kills both stem cells and developing spermatogonia; 30
LR Bucci and ML Meistrich produced azoospermia by 56 days after treatment at doses above mg / kg bw, "Effect of busulfan on spermatogenesis in mice; cytotoxicity, sterility. Sex, sperm abnormalities, and dominant lethal mutations (Ef
fects of busulfan on murine spermatogenesis: cytotoxicity, sterility, sp
erm abnormalities, and dominant lethal mutations.) '', Radiation Researc
h 176: 259-68 [1987]).

The present invention provides spermatozoa that are highly effective for transferring both heterologous as well as allogeneic genes into the germ cells of animals, either in vitro or in vivo, and for producing transgenic vertebrates. Address the need for plastic transfection. The present technology also addresses the requirements for germline and stem cell line gene therapy in humans and other vertebrate species, including the need for superior methods of depleting untransfected male germ cells from the testes. This technique is of great value not only in producing transgenic animals in large species, but also in repairing genetic defects leading to male infertility. Male germ cells that have stably integrated DNA can be selected.

[0018]   These and other advantages and features of the invention are described herein.

SUMMARY OF THE INVENTION The present invention relates to in vivo and ex vivo (in vitro) transfection of eukaryotic germ cells with desired genetic material. Briefly, in vivo methods involve direct injection of genetic material with a suitable vector into the testes of animals. In this method, all or part of the male germ cells in the testis are transfected in situ under effective conditions. The ex vivo method uses a novel isolation method to extract germ cells from the gonads of suitable donors or from the gonads of the animals themselves, transfect them in vitro or otherwise genetically modify them, and then spontaneously. Including returning them to the testes under suitable conditions to reestablish colonies. The ex vivo method has the advantage that the transfected germ cells may be screened by various means before they are returned to the testes to ensure that the transgenes are stably integrated into the genome. Moreover, after screening and cell sorting, only germ cell-rich populations may be recovered. This approach increases the probability of transgenic offspring after mating.

The present invention also provides a novel method for isolating spermatogonia, which comprises obtaining spermatogonia from a mixed population of testis cells by extruding the cells from the seminiferous tubules and dissociating them by mild enzymatic treatment. It also relates to the method. Genetically modified spermatogonia or stem cells are promoter sequences that are active only when rotating the cell cycle of a spermatogonial stem cell population, such as B-Myb or c-kit promoter region, c-raf-1 promoter, ATM
(Ataxia-telangiectasia) promoter, RBM (ribosome binding motif) promoter, DAZ (deleted in azoospermia) promoter, XRCC-1 promoter, HSP 90 (heat shock gene) promoter, cyclin A1 promoter , Or a spermatogonia-specific promoter, such as the FRMI (from the fragile X site) promoter, selectively with a reporter construct, eg, green fluorescent protein ([GFP] or enhanced GFP [EGFP]), yellow fluorescent protein, blue By a novel method comprising operably linking to and utilizing a fluorescent protein, a phycobiliprotein such as phycoerythrin or phycocyanin, or another protein that fluoresces under light of a suitable wavelength. It may be isolated from a mixed cell population. These unique promoter sequences drive expression of the reporter construct only when the spermatogonial cell cycle is rotated. Thus, spermatogonia are the only cells in the mixed population that express the reporter construct and may thus be isolated on this basis. Transgenic cells expressing fluorescent or luminescent reporter constructs can be sorted, for example, with the aid of a Flow Activated Cell Sorter (FACS) set at an appropriate wavelength, or they can be selected by chemical methods. Good.

The present invention also relates to an effective method of substantially depleting male germ cells from a vertebrate testis. The method reduces the dose of an alkylating agent such as busulfan and the dose of gamma irradiation to a vertebrate to substantially decellularize the vertebrate testis,
For example, it may include co-administration of sufficient amounts to prepare male germ cells from a donor animal for transplantation. With such a combination treatment of alkylating agent and gamma irradiation, when compared to either the alkylating agent or gamma irradiation alone,
Histologically superior results are obtained in eliminating the original population of untransfected or genetically unmodified male germ cells. Thus, this method of depleting vertebrate testicular cells maximizes the production of transgenic animals using the in vitro method of incorporating a polynucleotide encoding a desired trait or product into the mating male germ cell. Become.

The present invention also relates to reestablishment of the testes by germ cells isolated from fresh or frozen testis biopsies. These germ cells may or may not be genetically engineered prior to transplantation into the recipient testis.

Regarding transfection (ie gene transfer), the method of the invention comprises
Administering a composition comprising an amount of nucleic acid comprising a polynucleotide encoding a desired trait or product to a germ cell in an animal or in vitro. In addition, the composition includes an associated regulated promoter region composed of, for example, a nucleotide sequence. This is combined, for example, with retroviral vectors, adenovirus and adenovirus-related vectors, or gene delivery systems containing cell transfection facilitating agents such as liposome reagents or other reagents used in gene therapy. These were introduced under conditions effective to transport the nucleic acid segment into the germ cells of an animal, with a polynucleotide that is selectively inserted into the germ cell genome. Following DNA integration, the treated animals are either naturally mated or bred with the aid of assisted reproduction techniques to select offspring with the desired trait.

This technique is applicable to the generation of transgenic animals used as animal models, and to the modification of the genome of animals, including humans, by the addition, modification or subtraction of genetic material, which often leads to phenotypic changes. The method is also applicable when a person carries a gene for a recessive or dominant gene disorder, or when a person has a tendency to transmit a polygenic disorder to their offspring and changes the carrier status of animals, including humans. It is possible.

Formulations suitable for use with the method include a polynucleotide segment encoding the desired trait and a transfection facilitating agent, and an uptake facilitating agent, optionally with a substance that selectively protects against DNA cleavage. .. The different components of the transfection composition are also provided in the form of a kit, the components being included in a fixed dose in two or more different containers. Kits generally include different components in different containers. The other components are provided with the carrier in the kit.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention arose from the inventor's desire to improve existing methods for genetic modification of animal germ cells and production of transgenic animals. Existing methods are direct injection of DNA into zygotes produced in vitro or in vivo, or production of chimeric embryos using fetal stem cells incorporated into recipient undifferentiated embryo cells. . The embryos thus treated are then transplanted into the pre-stimulated uterus or fallopian tube. The currently available methods are very time consuming and costly, have to go through several invasive steps, and the production of transgenic offspring is accidental and unpredictable.

The inventors of the present invention have sought a less costly, faster, and more efficient procedure for obtaining transgenic animals in which a male animal carrying a nucleic acid fraction encoding the desired trait We have devised our method of transfecting germ cells in vivo or ex vivo. This method is based on at least one of the following strategies. The first method involves the delivery of nucleic acid fractions to the testes of animals using a known gene delivery system in situ (in vivo transfection), in which the transfected germ cells differentiate in their own environment. can do. Then, an animal (transgenic animal) in which it is known that the nucleic acid has been incorporated into germ cells is selected. The animals thus selected can produce transgenic offspring by mating, semen injection or in vitro fertilization using the sperm. Animals are selected after biopsy of one or both testes, or after amplification of the injected sperm of the animals by the polymerase chain reaction to confirm that the desired nucleic acid sequence has been incorporated. Gene encoding green fluorescent protein (or encoding enhanced green fluorescent protein [EGFP]) during initial transfection or gene delivery to facilitate confirmation of whether the desired nucleic acid was actually incorporated Alternatively, a gene encoding a yellow fluorescent protein, a blue fluorescent protein, a phycobiliprotein such as phycoerythrin or phycocyanin, or any other protein that fluoresces at an appropriate wavelength, or a gene encoding a photoprotein, etc. A defective reporter gene can be included.

Alternatively, male germ cells can be isolated from the donor animal and transfected or genetically modified in vitro to retain the desired trait. After such genetic manipulation, germ cells that show some evidence that the DNA has been modified as desired are selected and transferred into the testes of an appropriate recipient animal. Further selection can be performed after biopsy of one or both testes, or after the ejaculated semen of an animal has been amplified by the polymerase chain reaction to confirm that the desired nucleic acid sequence has been incorporated. As described above, upon initial transfection or gene delivery, it encodes green fluorescent protein (or enhanced green fluorescent protein [
EGFP] or a phycobiliprotein such as yellow fluorescent protein, blue fluorescent protein, phycoerythrin or phycocyanin, or any other protein that fluoresces under a light source of the appropriate wavelength, or luminescence A co-transfected reporter gene, such as a gene encoding a protein, can be included.

Another desirable reporter gene suitable for certain applications is a gene encoding a protein capable of enzymatically releasing light from a substrate; such a gene suitable for the purposes of this invention. The protein is a “photoprotein”. For example,
Examples of photoproteins include luciferase and apoaequorin.

Prior to transplantation of germ cells, the testes of the recipient animal may be tested in several ways in order to inactivate or destroy the endogenous germ cells, ie, to kill the testes of the endogenous male germ cells. It is common to process by one of them, or a combination thereof. This is done by any suitable means such as gamma irradiation, chemical treatment, methods using infectious agents such as viruses, depletion by autoimmunity, or a combination thereof. By extinguishing endogenous male germ cells, genetically modified donor cells can be grown in the testes of recipient animals.

Whatever means is used to extinguish the testes of endogenous male germ cells, they should not destroy the basic precision structure of the testis or cause serious damage. If the minute system of seminiferous tube formation is disrupted, exogenous spermatogonia may not be able to repopulate in the testis. Moreover, it is expected that the disruption of seminiferous tubules impairs the transportation of semen and makes it impossible to reproduce. In addition, this kind of slight testicular injury must be avoided so that it does not cause irreversible damage to the Sertoli cells. This is because these cells are the basis for their development during germ cell maturation. in addition,
It may also play a role in preventing the destruction of transplanted exogenous spermatogonia by the host's immune defense system.

More preferably, in extinct the testes of male germ cells resident in the recipient animal,
It is preferable to use a method for radically extinguishing the testes of vertebrates to which this invention relates. The method of the present invention for radically extinct vertebrate testis includes busulfan (1,4-butanediol dimethanesulfonate; Myle
ran, Glaxo Wellcome), chlorambucil, cyclophosphamide, melphalan, or a cytotoxic alkylating agent such as, but not limited to, ethylethanesulfonic acid and gamma irradiation in either order. It is meant to be used together. The combined use of alkylating agents and gamma irradiation gives very good results in killing the testes of germ cells compared to either administration alone. The dose of alkylating agent and the dose of gamma irradiation should be sufficient to fundamentally kill the testes.

A preferred dose of alkylating agent is about 4-10 mg / kg body weight,
Most preferred is about 6-8 mg / kg body weight. The administration of the alkylating agent may be a pharmaceutical formulation including, but not limited to, intraperitoneal, intravenous or intramuscular injection, intravenous drip, implantation, transdermal or transmucosal delivery system. It can be done by any delivery system that is acceptable to.

It is not essential to provide a recovery period between administration of the alkylating agent and irradiation, and it is most desirable that these two types of treatment be performed within 0 to 24 hours after the end of either treatment. Desirably, the interval between the two treatments should not exceed 2 weeks. The reason for this is that after 2 weeks, the best results cannot be obtained for the purpose of transplanting genetically modified or heterologous male germ cells into the testes of a recipient animal.

For the recipient vertebrate, about 200-800 Rad, most preferably about 350-450 Rad
Irradiate the testis locally with a dose of gamma rays to extinct it. Below 200 Rad, there is little effect, and above 800 Rad, symptoms of radiation sickness, especially gastrointestinal tract symptoms, usually occur. Male germ cells can be transplanted into an animal within 3 days to 2 months of being treated according to the method of the present invention for extinction of the testes of the recipient animal, as described above. Before 3 days, trace amounts of cytotoxic alkylating agents or endogenous apoptotic signal molecules may remain in the recipient animal, damaging the transplanted male germ cells. After 2 months, male germ cells within the body usually begin to regenerate, and transfected, genetically modified, or xenogeneic male germ cells are transplanted into the testes of recipient animals for mating purposes. In case,
You can't get the best results.

Animals that have been shown to have appropriately modified sperm cells can be mated naturally or the spermatogonia can be used for semen injection or in vitro fertilization. The transgenic offspring thus obtained can be bred to obtain further transgenic offspring by means of either natural mating or artificial semen injection. The method of the present invention requires less number of invasive procedures than other methods currently used, and has a high success rate in incorporating a nucleic acid sequence encoding a desired trait into the genome of progeny.

Primordial germ cells are believed to develop from the ectoderm of the embryo and are first found in the epithelium of the E8 stage endoderm yolk sac. These cells travel from there via the hindgut endoderm to the genital ridge. Primitive spermatogonial stem cells, known as A0 / As, differentiate to B
Become type spermatogonia. This type B spermatogonia further differentiates to form primordial spermatogonia, which undergoes long-term meiosis to undergo homologous chromosome pairing and recombination. There are several distinct morphological steps in meiosis: pre-filamentary stage, fine-filamentary stage, mitotic stage, thick-line stage,
The second spermatocyte, and the haploid sperm cell. Haploid spermatids undergo further morphological changes, including nuclear shape changes, acrosome formation, and tail assembly during spermatogenesis. The final changes in sperm occur in the female reproductive tract prior to fertilization. The nucleic acid fraction administered into the gonads by the in vivo method of the invention reaches the germ cells at one or more of these phases and is taken up by cells in the more sensitive phases. In the case of ex vivo (in vitro) genetic modification methods, it is common to use only diploid spermatogonia for nucleic acid modification. The cells are modified in vivo using gene therapy techniques or in vitro using various transfection techniques.

[0038] Thus, the present invention provides for the isolation of male germ cells in the present invention and for the treatment of allogeneic and heterologous DNAs in vivo and ex vivo (in vitro).
It provides a new and very clear method for transfecting (or gene delivery) into germ cells of animals. That is, a preparation comprising a gene delivery system and at least one nucleic acid fraction is administered to an animal in an amount and under conditions effective to modify the germ cells of the animal, and the nucleic acid fraction is released into the germ cells and released. It is a method of being integrated into the genome.

One method of introducing the mixture for gene delivery into germ cells in vivo is by direct delivery into the testes of animals, which are distributed in the male germ cells at each stage of development in the testes. It The in vivo method utilizes new techniques such as injecting the mixture for gene delivery into the efferent tubule, into the seminiferous tubule, or into the testicular reticulum, eg, using a micropipette. Pico pumps capable of delivering precise volumes under controlled pressure values to ensure that the mixture for gene delivery is infused under pressure that does not damage the delicate tubule system in the testis It is advisable to inject it through a micropipette using The micropipette may be made of a suitable material such as metal or glass, and is generally composed of, for example, a glass tube that is passed through a small hole at the tip of the operation using a pipette puller. The tip may be angled in a convenient manner to facilitate entry into the testicular tubule system. The micropipette may also have a tilted operating tip for better and less damaging seminiferous puncture at the injection site. This tilt can be created by specially manufactured polishing equipment. The diameter of the tip of the pipette used for in vivo injection method is about 15
~ 45 microns, but other sizes can be used as needed depending on the size of the animal. The tip of the pipette should be used with the aid of a coaxial-illuminated binocular microscope, taking care not to damage the seminiferous tubule wall opposite the injection site and to minimize trauma. Introduced into the seminiferous tube system. On average, a bench mount micromanipulator is not necessary, as a magnification of about 25 to 80 times is suitable and this procedure is performed by a skilled technician without the help of others. A small amount of a suitable non-toxic dye may be added to the gene delivery solution to confirm delivery and distribution of the testes into the seminiferous tubules. That is, such as a suitable non-toxic dilute solution of the dye that can be viewed and traced under a microscope.

In this way, the mixture for gene delivery is brought into direct contact with germ cells. Generally, the mixture for gene delivery will include a modified nucleic acid encoding the desired trait in addition to a suitable promoter sequence, and a liposome, retroviral vector, adenovirus vector, adenovirus enhanced gene delivery system, or a combination thereof. It is composed of any agent that increases the uptake of nucleic acid sequences. A reporter construct, such as a gene encoding green fluorescent protein, may also be added to the gene delivery mixture. Targeting molecules such as c-kit ligands can also be added to the gene delivery mixture to facilitate transfer of male germ cells.

With respect to the ex vivo (in vitro) method of genetic modification, introduction of modified germ cells into the recipient testis may be performed by direct injection with a suitable micropipette. Supporting cells such as Leydig or Sertoli cells, which provide hormonal stimulation for spermatogonia differentiation, may be transferred to the recipient testis along with the modified germ cells. These transfected feeder cells may be unmodified, or may themselves be co-transfected with or separately from germ cells. These transferred feeder cells may be autologous or heterologous to either the donor or recipient testis. The preferred concentration of cells in the transfer fluid may be easily established by simple experimentation, but is approximately 1 x 10 ⁵ to 10 x 10 ⁵ cells / solution 10 µ.
It will be in the range of l. The micropipette may be introduced into the efferent tubing, testicular net, or seminiferous tubing with the help of picopumps to selectively control pressure and / or volume, or this transport may be done manually . The micropipette used is similar in most respects to that used for in vivo injection, except that the tip diameter is approximately 70 microns. The microsurgical method of introduction is similar in all respects to that used in the in vivo method described above. Suitable dyes may be incorporated into the carrier solution so that satisfactory translocation of the transfected germ cells can be readily identified.

The transfected germ cells preferably transfer to the testes of the recipient animal, although the recipient animal can, but need not be, the same donor animal. The testes of the donor animal are preferably depleted of native germ cells prior to transfer of the transfected germ cells therein. This cell loss can be done by any suitable means. However, the vertebrate testis is most preferably cytopathiZed by co-treatment of the animal With an alkylating agent and gamma irradiation according to the present method of substantially depleting vertebrate testis cells. Donor male germ cells can then be transferred to recipient mice.

Upon contact with germ cells, whether in situ or in vitro in animals, the gene transfer mixture allows incorporation of the heterologous genetic material into the appropriate cell location so that it is integrated into the genome and expressed. And facilitate transportation. Nucleic acid sequences may be incorporated into cells using a number of known gene transfer methods.

“Gene transfer (or transfection) mixture” within the meaning of this patent
By, for example, is meant the genetic material selected with the appropriate vector mixed with an effective amount of lipid transfectant. The amount of each component of the mixture is chosen to optimize transfection and genetic modification of the particular species of germ cell. Such optimization requires only routine experimentation. The ratio of DNA to lipid is wide, preferably about 1: 1, but the lipid material and D used
Other ratios may be used depending on the type of NA. This ratio is not important.

As used herein, “transfectant” refers to a genetic material that enhances the uptake of exogenous DNA segments into eukaryotic cells, preferably mammalian cells, and more preferably mammalian germ cells. Means important composition of matter. Enhancement is measured relative to uptake in the absence of transfection agent. Examples of transfection agents include adenovirus transferrin polylysine DNA complex. These complexes generally enhance DNA uptake into cells and reduce cleavage during transit from the cytoplasm to the nucleus of cells. These complexes may be targeted to male germ cells with specific ligands recognized by receptors on the cell surface of germ cells, such as the c-kit ligand or modifications thereof.

Other preferred transfection agents include lipofectin, lipofectamine,
Includes DIMRIE C, Superfect, and Effectin (Qiagen).
Although these are not as effective transfectants as viral transfectants, they do not have the size limitations commonly associated with virus-derived transfectants and allow stable integration of heterologous DNA sequences into the vertebrate genome. Has the advantage of promoting

As used herein, the term “virus” means any virus, or transfected fragment thereof, that may facilitate the transfer of genetic material to male germ cells. Examples of suitable viruses herein include adenovirus, adeno-associated virus, retroviruses such as human immunodeficiency virus, lentiviruses such as Moloney murine leukemia virus, and vesicular stomatitis virus glycoprotein (VSV-G). -A retroviral vector derived from the Moloney virus called Moloney murine leukemia virus, a mumps virus, and a transfected fragment of any of these viruses, and uptake and release of the desired DNA segment by the germ cell cytoplasm. Other viral DNA segments that promote, as well as mixtures thereof. Mumps virus is particularly suitable due to its affinity for immature sperm cells, including spermatogonia. All of the above viruses may require modification to render them avirulent or less antigenic. However, it is within the scope of the invention to use other known vector systems.

As used herein, “genetic material” refers to novel genetic modification (s),
Or, a DNA sequence capable of conferring biologically functional characteristic (s) on a recipient animal. The novel genetic modification (s) or characteristic (s) may be encoded by one or more genes or gene segments, or
It may be caused by the removal or mutation of one or more genes and may further include regulatory sequences. The transfected genetic material is preferably functional, ie they express the desired trait either by the product or by suppressing the production of another. An example of another mechanism of action by which the function of a gene is expressed is genomic imprinting, in which one of a pair of genes (alleles) is inactivated during early embryonic development, or Inactivation of genetic material by mutations or deletions, or, among other things, suppression of dominant negative gene products.

Furthermore, the novel genetic modification (s) may be an artificially induced mutation or variant, or a naturally occurring allelic variant or genetic variant. Mutations or variants may be artificially induced by many techniques, all well known in the art, such as chemotherapy, gamma irradiation treatment, UV irradiation treatment, UV irradiation and the like. Chemicals useful in inducing mutations or variants include carcinogens such as ethidium bromide, and others known in the art.

A DNA segment of a particular sequence may be constructed so that it incorporates the desired mutation or variant, or disrupts the gene or alters the genomic DNA. One of ordinary skill in the art will readily recognize that the genetic material is heritable and therefore present in nearly every cell of a future generation of progeny, including germ cells. A novel feature is expression of a previously unexpressed trait, enhancement or reduction of an expressed trait, overexpression or underexpression of a trait, ectopic expression, i. It is the attenuation or disappearance of the traits. Other novel features include qualitative alterations in the expressed traits, eg, to reduce or alleviate or otherwise prevent the development of inherited disorders with a polygenic basis.

In order to express the transfected or otherwise transported genetic material in order to obtain the desired trait, the promoter sequence is functionally linked to the polynucleotide sequence encoding the desired trait or product. Are connected. For purposes of this invention, "operably linked" means that the promoter sequence is located upstream of the coding sequence and that transcription is in vitro of all essential enzymes, transcription factors, mirror factors, activators and It is meant that both sequences are in the 5'to 3'direction, as occurs in the presence of reactants under convenient physical conditions, such as a suitable pH and temperature. This does not mean that the conditions favor transcription in any particular cell.

Select a promoter sequence that is functional in the cell type of interest. A promoter sequence that is only active in rotating the cell cycle of the spermatogonial stem cell population, eg
B-Myb or c-kit promoter region, c-raf-1 promoter, ATM (Ataxia-
Telangiectasia) promoter, RBM (ribosome binding motif) promoter, DAZ (deleted in azoospermia) promoter, XRCC-1 promoter, H
SP 90 (heat shock gene) promoter, cyclin A1 promoter, or F
A spermatogonia-specific promoter, such as the RMI (from the fragile X site) promoter,
It can be used for differential expression in male germ cells.

The human cyclin A1 promoter region, non-human homologue or functional fragment thereof is
Where expression is desired in vertebrate germ cells, hematopoietic cells, or other stem cells, the most preferred promoter sequence to drive expression of the reporter construct or to drive expression of another desired heterologous gene sequence. (C. Muller et al., J. Biol. Chem. 274 (16): 11220-28 [April 16, 1991]).

The present invention also relates to kits for transfecting or otherwise genetically modifying male vertebrate germ cells useful for obtaining transgenic male germ cells. The kit is a ready-made collection of materials to facilitate transfection or genetic modification of vertebrate male germ cells. The kit of the present invention comprises a transfection (or gene transfer) substance as described above, and an alkylating agent used for substantially depleting a vertebrate testis according to the method for cell loss of a vertebrate testis. Included with instructions for effective use.

Optionally, the kit includes a radiation shield capable of specifically directing gamma irradiation to the testis. The shield comprises a lead and another dense material that tends to absorb gamma radiation, the shield being a slot, hole, tube or other suitable for selectively directing gamma radiation to the testes of a male vertebrate. Including the means.

Optionally, the kit comprises a polynucleotide comprising a promoter sequence operably linked to a DNA sequence encoding a reporter gene, preferably a fluorescent or light emitting protein as described above. Optionally, the kit comprises an immunosuppressant such as cyclosporine or corticosteroids and / or further nucleotide sequences encoding the expression of the desired trait. The materials or components included in the kit are
Any convenient and suitable method that preserves its functionality and utility is preserved and provided to the practitioner. For example, the ingredients can be in solution, dehydrated, or lyophilized; they can be provided at room temperature, refrigerated, or frozen.

The present invention also relates to a method of isolating spermatogonia, which comprises obtaining spermatogonia from a mixed population of testis cells by extruding the cells from the seminiferous tubules and dissociating them by mild enzymatic treatment. . Genetically modified spermatogonia or stem cells are promoter sequences that are active only in the case of stem cells, such as the human cyclin A1 promoter,
Alternatively, when initiating the cell cycle of the spermatogonial stem cell population, for example, B-Myb or c
-kit promoter region, c-raf-1 promoter, ATM (ataxia-telangiectasia) promoter, RBM (ribosome binding motif) promoter, DAZ (deleted in azoospermia) promoter, XRCC-1 promoter , A spermatogonia-specific promoter, such as the HSP 90 (heat shock gene) promoter, or the FRMI (from the fragile X site) promoter, eg, green fluorescent protein (or EGFP), yellow fluorescent protein, blue fluorescent Contains a gene encoding a protein, a phycobiliprotein such as phycoerythrin or phycocyanin, or other protein that fluoresces under light of a suitable wavelength, or a light-emitting protein such as luciferase or apoaequorin By a novel method, which involves using it in combination with a construct. From a mixed population of cells may be isolated. These unique promoter sequences drive expression of the reporter construct only when the spermatogonial cell cycle is rotated. Thus, spermatogonia are the only cells that express the reporter construct (s) in the mixed population and may thus be isolated on this basis. In the case of fluorescent reporter constructs, the cells may be sorted, for example with the aid of FACS set to the appropriate wavelength (s), or they may be selected by chemical methods.

The method of the present invention is suitable for a variety of vertebrate applications, all of which are capable of producing gametes, ie sperms or eggs. Thus, according to the present invention,
The novel genetic modification (s) and / or characteristic (s) can be expressed in humans, non-human primates, eg farm animals such as monkeys, marmosets, sheep, cows, pigs, horses, especially racehorses, marine mammals, wild animals. It may be given to animals, including animals, rodents such as mice and rats, mammals such as gerbils, hamsters, rabbits and the like. Other animals include birds such as chickens, turkeys, ducks, ostriches, emu, geese, guinea fowl, pigeons, quail, rare and ornamental birds and the like. Of particular importance are endangered species of wildlife such as rhinoceros, tiger, cheetah and condor specific species.

A “transgenic” animal in the broadest sense is an animal in which foreign DNA has been permanently introduced into its cells. The foreign gene (s) introduced into the animal's cells are called the "transgene (s)". The present invention comprises a heterologous gene comprising biologically functional genetic material, ie, exogenous transgenic genetic material or material from a different species, in its native undisturbed form as it exists in animal germ cells. It can be applied to the production of transgenic animals. In another example, the genetic material is "allogeneic" genetic material obtained from different strains of the same species, eg, "normal" forms of the gene, or animals carrying the desired alleles thereof. Similarly, the gene
It may be a hybrid construct containing a promoter DNA sequence and a DNA coding sequence linked to each other. These sequences may be obtained from different species, or they may be normally non-contiguous DNA sequences from the same species. The DNA construct may also include DNA sequences from prokaryotes such as bacteria or viruses.

In one preferred embodiment, the transfected germ cells of the transgenic animal have incorporated non-endogenous (exogenous) genetic material into their chromosomes. This is called "stable transfection". One of ordinary skill in the art will readily recognize that any desired trait resulting from a change to the genetic material of any transgenic animal produced by the present invention can be inherited. The genetic material is initially inserted only in the germ cells of the parent animal, but it will eventually be present in future offspring and later generation germ cells. Genetic material is also present in the differentiated cells of the progeny, the somatic cells. The invention also includes progeny obtained from a cross of the transgenic animal. Transgenic animals crossed with other transgenic or non-transgenic animals of the same species will produce some transgenic fertile offspring. Thus, the present invention provides for the breeding of the transgenic animal (s) provided herein, as well as the animal strain (s) resulting from the breeding of its fertile offspring.

“Mating” within the meaning of this patent means mating male and female animals such that fertilization occurs. Such mating may be obtained by natural mating, ie mating, or by in vitro or in vivo artificial means. Artificial means include, but are not limited to, artificial insemination, in vitro fertilization, cloning and embryo transfer, intracytoplasmic spermatogonia microinjection, cloning and embryo division. However, other ones may be used.

Transfection of mature male germ cells can also be performed utilizing the present technique in isolating cells from vertebrates as is known in the art and as shown in Example 10. Good. The cells thus isolated are ex vivo (in vitro)
Or may be cryopreserved as described in Example 11. The actual transsection of the isolated testis tissue may be performed, for example, by isolating the vertebrate testes, removing the coating, and tearing to chop the vas deferens. It is then known to loosely cleave the tissue matrix, such as bovine serum albumin and modified DMEM medium, with pancreatic trypsin, type I collagenase, type I pancreatic DNase to release undamaged cells. The dissociated cells may be incubated in an enzyme mixture containing the specified enzyme. 5 cells in the enzyme mixture
Minutes to about 30 minutes, more preferably about 15 to about 20 minutes, may be incubated at a temperature of about 33 ° C to about 37 ° C, more preferably about 36 to 37 ° C. After washing the enzyme mixture from the cells,
They may be placed in an incubation medium such as DMEM and seeded on a culture dish. Many commercially available transfection mixtures may also be mixed with the polynucleotide encoding the desired trait or product for transfection of cells. The transfection mixture is then mixed with the cells for about 2 hours to about 16 hours, preferably about 3 hours to 4 hours at a temperature of about 33 ° C to about 37 ° C, preferably about 36 ° C to about 36 ° C.
The interactions may be at 37 ° C., and more preferably in a constant and / or controlled atmosphere. After this period, the cells are preferably left at a lower temperature of about 33 ° C to about 34 ° C, preferably about 30-35 ° C for about 4 hours to about 20 hours, preferably about 16-18 hours. Other conditions that do not deviate significantly from what is described may also be utilized as known to those skilled in the art.

The method is applicable in the field of gene therapy because it allows the introduction of genetic material that encodes and regulates specific genetic traits. Thus, in humans, for example,
Treating a parent may correct many monogenic disorders that may otherwise affect the child. Similarly, altered expression of a complete or at least partially genetic basis caused by the interaction of one or more genes, or a disorder that is more common due to the involvement of multiple genes. It is also possible to let. This technique may be applied in a similar manner to treat disorders in animals other than human primates. In some cases, where multiple genes are involved in the expression or suppression of defined traits, it may be necessary to introduce one or more "gene (s)" into the germ cells of the animal in order to obtain the desired therapeutic effect. There may be In humans, examples of polygenic disorders include diabetes mellitus, inflammatory bowel disease, certain types of atherosclerotic cardiovascular disease and hypertension, schizophrenia and schizophrenia caused by lack of production of insulin or a response to insulin. Types of chronic depression are included. In some cases, one gene may encode an expressible product and another gene may encode a regulatory function, as is known in the art. Other examples are point mutations or depletion of the genome, inactivation of genes causing disease or disease, or insertion of genes that are dominant negatively expressed, or regulation such as gene promoters, enhancers, untranslated tail regions of genes. This is the case when applying the homologous recombination method in order to repair the alteration of the element or the regulation of the extension of the DNA repeat sequence which causes diseases such as Huntington's disease, fragile X-syndrome and the like.

A particular reproductive application of this method is in the treatment of animals, especially humans with impaired spermatogenesis. Defects in sperm completion or spermatogenesis often have a genetic basis, ie, one or more mutations in the genome can result in defective production of normal sperm cells. This can occur at various stages of germ cell development, and infertility in men (
Infertility or sterility may occur. The present invention is applicable to the establishment of spermatogenesis, for example to insert or integrate nucleic acid sequences into the genome of a recipient, thereby treating hypospermia or azoospermia in the treatment of infertility. Similarly, the method is applicable to men who have hypofertility or infertility due to movement disorders that have a genetic basis.

The method further applies to the production of transgenic animals which express substances which are therapeutically useful for use in human and veterinary medicine or for the sake of well-being.
Examples include the production in milk of agents such as factors that enhance blood coagulation in hemophilia type patients, or hormonal substances such as insulin and other peptide hormones.

The method also applies to the generation of transgenic animals of anatomical and physiological phenotype suitable for human xenograft transplantation. With transgenic technology,
It allows the production of animals that are immunologically compatible with human recipients. For example,
Appropriate organs may be removed from such animals and transplanted, for example, to the heart, lungs, and kidneys.

In addition, the germ cells transfected according to the present invention may be extracted from transgenic animals and stored under conditions effective for later use, as is known in the art. Storage conditions include the use of programmed freezing and / or cryopreservation with cryoprotectants, and the use of storage in substances such as liquid nitrogen. Germ cells may be obtained in the form of whole biopsies of male animal sperm, or isolated sperm or immature spermatocytes, or testis tissue containing primordial germ cells. Such preservation techniques are particularly useful for young human adults or children who are seeking tumor treatment for diseases such as leukemia or Hodgkin lymphoma. These treatments often cause irreversible damage to the testes, thus rendering spermatogenesis incapable of treatment, for example, after treatment with radiation or chemotherapy. If germ cells are preserved and later transferred to the testes, fertility can be restored. In such situations, transfection and manipulation of germ cells will occur as taught in the present invention, but transfection is generally not appropriate or necessary.

In non-human species, the technology is valuable for transporting gametes as frozen germ cells. Such transportation facilitates colonization of various valuable livestock or birds that are remote from the donor animal. This approach also applies to the conservation of endangered species across the globe.

The invention will be described in more detail with reference to the following non-limiting examples. All references and relevant portions of the published patent applications cited in this patent that are necessary to achieve the purpose are incorporated herein by reference.

Examples In Vivo and In Vitro Transfection of Male Embryo Cells In Vivo Adenovirus Enhanced Transferrin Polylysine Mediated Delivery of Green Lanthanum Reported Gene Delivery System to Testis Cells
Curiel et al. (Curiel DT et al., Adenovirus Enhancement of Transferrin Polylysine-Mediated Gene Delivery, PNAS USA 88: 8850-8854 (1991)). DNA release is dependent on transferrin receptor-mediated endocytosis (Wagner et al., Transferrin polycation conjugate as a carrier for DNA uptake into cells). This method also relies on the ability of the adenovirus to disrupt cellular vesicles such as endosomes and release the entrapped contents. This system can enhance gene delivery to mammalian cells by as much as 2000-fold over other methods.

The gene delivery system used in the in vivo experiments was constructed as shown in the examples below.

Example 1 Preparation of Transferrin-Poly L-Lysine Complex Human transferrin was prepared by using EDC (1-ethyl-3- (3-dimethylaminopropyl carbodiimide hydrochloride) according to the method of Gabarek and Gergely (Gabare
k & Gergely, Zero-Length Cross-Linking Procedure Using Active Esters, Analyt. Biochem 185
: 131 (1990))) conjugated to poly (L-lysine). In this reaction, EDC reacted with the carboxyl group of human transferrin to form an amino-reactive intermediate.
The activated protein was reacted with poly (L-lysine) moieties for 2 hours at room temperature and the reaction was stopped by the addition of hydroxylamine at a final concentration of 10 mM. The conjugate was purified by gel filtration and stored at 20 ° C.

Example 2 Generation of DNA for In Vivo Transferrin The green lanthanum-1 vector (Life Technologies, Gibco BRL, Geisersburg, Md.) Is used to monitor gene transferrin in mammalian cells. It is a reporting structure. It consists of a gene encoding green fluorescent protein (GFP) triggered by the immediate early promoter of cytomegalovirus (CMV). The downstream part of the gene is the SV40 polyadenylation signal. When illuminated with blue light, cells were transferrin with green lanthanum-1 fluorescence along with bright green light. The excitation peak value was 490 nm.

Example 3 Generation of Adenovirus Particles A replication-incompetent adenovirus dI312 in which the Ela portion was deleted was produced by Jones and She.
Proliferated in Ela trans-complementing cell line 293 as described by nk (Jones and S
henk, PNAS USA (1979) 79: 3665-3669). Mitt prepares for a large-scale virus
ereder and Trapnell method (Mittereder et al., "Evaluation of adenovirus vector concentration and biological activity for gene therapy" J. Urology, 70: 7498-7509 (1996).
)). The virion concentration was examined by UV spectroscopy, one extinction unit equal to 10 viral particles / ml. The purified virus was stored at -70 ° C.

Example 4 Formation of Transferrin-Poly L-Lysine DNA Virus Complex 6 μg transferrin-polylysine complex from Example 1 was prepared in Example 7.3
x 10 ⁷ and then mixed with 5 μg green lanthanum DNA construct of Example 2 and left at room temperature for 1 hour. About 100 μl of the mixed solution was taken with a micropipette, placed on a pipette extractor, and slightly bent on a microforge. Solution-filled micropipettes were attached to picopumps (Ependorff) and DNA complexes were delivered to mice under continuous pressure in vivo as described in Example 6. Controls were performed by the same procedure but omitting the transferrin-polylysine-DNA virus complex from the dosing mix.

Example 5: Efficiency comparison of adenovirus-enhanced transferrin-polylysine and lipofectin-mediated transferrin DNA conjugated complexed adenoeurus particles were tested in vitro in vitro prior to in vivo testing.
Tested on HO cells. In these experiments, the luciferase report gene was used for ease of quantifying luciferase activity. The expression construct consists of the reported gene-encoded luciferase and is induced by the CMV promoter (Invitrogen, Carlsbad, CA 92008). CHO cells were Dul with 10% fetal bovine serum
Grow in becco's modified Eagle's medium (DMEM). In gene transfer experiments, CHO cells were seeded in 6 cm tissue culture plates and grown to approximately 50% confluency (5 x 10 ⁵ cells). Prior to transferrin, medium was aspirated and replaced with serum free DMEM. Cells were transferrin with transferrin-polylysine DNA complex or lipofectin DNA aggregates. For transferrin-polylysine DNA transfer, the DNA adenovirus complex was added to cells at a concentration of 0.05-3.2 × 10 ⁴ adenovirus particles per cell.
The plate was returned to the 5% CO ₂ incubator at 37 ° C for 1 hour. After 1 hour, 3 ml of complete) medium was added to the wells and the cells were cultured for 48 hours before harvesting. Cells were removed from the plates, counted and lysed for determination of luciferase activity.

For cells transferred with lipofectin, 1 μg of CMV luciferase DNA was incubated with 17 μl lipofectin (Life Technologies). DNA lipofectin aggregates were added to CHO cells and incubated for 4 hours at 37 ° C. with 5% CO ₂ . Three
ml of complete medium was added to the cells and cultured for 48 hours. Cells were harvested, counted and lysed for luciferase activity. Luciferase activity was measured with a luminometer. The obtained results are shown in Table 1.

The data included in Table 1 show that the adenovirus-enhanced transferrin-polylysine gene delivery system is 1,808 times more efficient for transferrin in CHO cells compared to lipofection.

[Table 1]

Example 6 In Vivo Delivery of DNA to Animal Germ Cells via the Transferrin-L-Lysine DNA Virus Complex The GFP DNA-transferrin-polylysine virus complex, prepared as described in Example 4, was It was released into the vas deferens of weekly B62F1 male mice. DNA delivery by transferrin receptor-mediated endocytosis is described by Schmidt et al. And Wagner et al. (Schmid
t et al., Cell 4: 41-51 (1986); Wagner E. et al. PNAS (1990), (USA) 81: 3410-3414 [1990]
). In addition, this delivery system relies on the ability of adenovirus to destroy cellular vesicles, such as endosomes, and release trapped contents.
The transferrin efficiency of this system is about 2,000 times higher than that of lipofection.

Male mice were anesthetized with 2% avertin (100% avertin is composed of 10 g of 2,2,2 tribromoethanol (Aldrich) and 10 ml of t-amyl alcohol (Sigma)) and a small incision made posteriorly. Going to the ventral skin and body wall at the level of the legs. The animal testes were pulled through the opening by grasping the testis fat pad with forceps. The seminiferous tubules were exposed and supported with a glass syringe. The GFP DNA-transferrin-polylysine virus complex is a glass syringe in your hand or an automatic pressure pipette (Eppendor).
Injection was made into a single vas deferens using a glass micropipette attached to f). Trypan blue was added to the seminiferous tubules to observe the entry of the mixed solution. The testis was then returned to the body cavity, the body wall was sutured, the skin was closed with a wound clip, and the animal was allowed to recover on a warm pad.

Example 7: Detection of DNA and Transcriptional Messages On day 9 post delivery of genetic material to the testes of animals, two animals were killed, the testes were removed, cut in half and frozen in liquid nitrogen. It was DNA from one half of the tissue and RNA from the other half were extracted and analyzed.

(A) DNA Detection The presence of GFP DNA in the extracts was examined 9 days after administration of the transferrin mixture using the polymerase chain reaction and GFP-specific oligonucleotides. GFP DNA was present in the testes of animals that received the DNA complex, but not in sham-operated animals.

(B) RNA Detection The presence of GFP mRNA was quantified in testis of experimental animals as follows. RNA was extracted from injected and non-injected testes and the presence of GFP message was detected using reverse transcriptase PCR (R TPCR) with GFP-specific primers. The GFP message was present in the injected testes but not in the control testes. Thus, the DNA detected by PCR analysis is effectively episomal GFP DNA or GFP DNA integrated into the animal's chromosome. The transferrin gene was expressed.

Example 8: Expression of Non-Endogenous DNA Two mice, one injected with the GFP-transferrin mixed solution injection and the other operated alone control, were sacrificed 4 days after injection, and the testes were excised. It was fixed with 4% paraformaldehyde at 18 ° C. for 18 hours. Fixed testes contained 2mM MgCl ₂ for 18 hours at 4 ° C
It was placed in 30% sucrose with PBS, embedded in dry ice frozen OCT, and sectioned. When the testes of both animals were examined by confocal microscopy with a fluorescent light of 488 nM wavelength, bright fluorescence was detected in the seminiferous tubules of GFP-injected mice but not in the control testes. A large number of cells in the vas deferens of GFP-injected mice showed bright fluorescence, which was evidence of the expression of fluorescent green protein.

Example 9: Offspring Generation from Normal Mating GFP transferrinized males were mated with normal females. The female became fully pregnant and gave birth. The offspring (F1 offspring or offspring) were examined for the presence of new genetic material.

Example 10: In vitro transferrin of testis cells. Cells were isolated from three 10 day old males. The testis was decapsulated, and the seminiferous tubules were finely torn and chopped with a sterile needle. The cells were incubated at 37 ° C. for 20 minutes in the enzyme mixed solution.
The enzyme mix consisted of 10 mg bovine serum albumin (tested germ), 50 mg bovine pancreatic trypsin type III, clostdium collagenase type I, 1 mg bovine pancreatic DNAse type I in 10 ml modified HTF medium. (Irvine Scientific, Irvine, CA). Enzymes were obtained from Sigma (St. Louis, MO 63178). After digestion, the cells were washed twice with HTF medium by centrifugation at 500 xg and resuspended in 250 µl HTF medium. Cells were counted and 0.5x10 ⁶ cells were plated on 60 mm culture dishes in a total volume of 5 ml DMEM (Gibco-BRL, Life Technologies, Geisesburg, MD 20884). Transferrin mixed solution was 5 μl Green Lantern DNA (Gibco
-BRL, Life Technologies, Gaithersburg, MD 20884) and 20 μl
Superfect (Qiagen, Santa Clarita, CA 91355) and 150
It was prepared by mixing μl DMEM. The transferrin mixture was added to the cells and allowed to warm at 37 ° C., 5% CO ₂ for 3 hours. Transfer the cells to a 33 ° C incubator,
Cultured overnight.

The next morning, cells were evaluated for transferrin efficiency by counting fluorescent cell numbers. In this experiment, transferrin efficiency was 90% (not shown). The green lanthanum transferrin testis cells found in Nomaski Optex x20 represent the same cells found in FITC. Almost all cells are fluorescent, which confirms the success of transferrin.

[0088] Example 11: Preparation of cell suspension from testis tissue for cryopreservation   Cell suspensions were prepared from mice of different ages as described below.     Group I: 7-10 days old     Group II: 15-17 days old     Group III: 24-26 days old

The mouse testis was dissected, placed in phosphate buffered saline (PBS), decapsulated and the seminiferous tubules separated. Seminiferous tubules from groups I and II were treated with HEPES buffered medium (D-D-) containing 1 mg / ml bovine serum albumin (BSA) (Sigma, St. Louis, MO 63178) and collagenase type I (Sigma) for the removal of ductal cells. MEM) (Gibco-BRL, Life Technolog
ies, Geyselsburg, MD). After 10 minutes of incubation at 33 ° C,
The seminiferous tubules were transferred to fresh medium. Digestion with this enzyme was not performed in Group I testis due to its brittleness.

Seminiferous tubules from Group II and III mice and whole tissue from Group I mice were transferred to Petri dishes containing medium and cut into 0.1-1 mm pieces using a sterile scalpel and needle. The minced tissue was centrifuged at 500 xg for 5 minutes and the pellet resuspended in 1 ml of enzyme mix. The enzyme mixture was prepared in D-DMEM together with HEPES (Gibco-BRL), and 1 mg / ml bovine serum albumin (
BSA) (Sigma, tested germ), 1 mg / ml collagenase I (Sigma), 5 mg / ml bovine pancreatic trypsin (Sigma), 0.1 mg / ml deoxyribonuclease I (DN-EP, Sigma)
). The seminiferous tubules were incubated at 33 ° C for 30 minutes in the enzyme mixture. After culturing,
1 ml of medium was washed twice in the medium by centrifugation and resuspension. After the final wash, the cell pellet was resuspended in 250 μl medium and counted.

Example 12: Transferrin Transferred Male Germ Cells to the Recipient Testes The cells were injected into the testes via the export tube using a micropipette. Total volume 50
3 × 10 ⁵ cells in μl were used for injection. Cells were mixed with trypan blue before injection.
Recipient mice were anesthetized with 0.017 mL / g body weight avertin. An incision was made across the bottom abdominal wall and the testis was gently pulled externally through the incision on the fat pad associated with the testis. The export tube was exposed and about 20 μl of cell suspension was injected into the export tube using a glass micropipette held in a steel micropipette holder (Leitz). The cells were forced out of the pipette by applying air pressure from a 20 mL glass syringe. Recipient testes reduced endogenous male germ cells prior to transfer of transferrin germ cells to the recipient animal.

Example 13: Reducing Treatment of Recipient Testis Male Germ Cells A comparison of reducing treatments. C57BL / 6J mice at 8 weeks of age were acclimatized for several days and assigned to the following three treatment groups. They were as follows: (1) 400 rad gamma irradiation, (2
) Busulfan combined with 4 μG / g body weight (1,4-butanediol dimethanesulfonate, Myleran, Glaxo Wellcome), and (3) Busulfan 400 rad gamma 1 week after irradiation (4 μg / g body weight). The fourth group of untreated C57BL / 6J, which was the same age as the treatment group, was used as a control. There were 24 mice in each treatment group, 5 hours after treatment,
At 24 hours, 48 hours, 72 hours, 1 week, 2 weeks, 1 month, 2 months, 3 animals were killed each.

In addition, other C57BL / 6J mice treated with the combined busulfan / 400 Rad treatment were histologically examined up to 5 months after treatment (testis of these mice was sectioned and H & E.
Prior to staining, it was fixed overnight in 4% paraformaldehyde in PBS at pH 7.4 and 4 ° C. ).

Administration of alkylate to the recipient spine. Busulfan-treated male mice
A dose of 4 μg busulfan / g body weight was administered. Busulfan is 100 immediately before injection
8 mg / mL was dissolved in% dimethylsulfoxide (DMSO) and diluted 1: 1 in phosphate buffered saline at pH 7.4. The diluted busulfan solution was injected intraperitoneally.

Radiation therapy of the recipient spine. For gamma irradiation treatment, mice were anesthetized with 0.017 mL / g body weight of 2.5% Avertin. Gamma irradiation was performed on the testes in the following manner. Each mouse was placed in a lead chamber with its testis and fundus exposed through an elliptical hole to the irradiation source ( ¹³⁷ Cs gamma cell 40 irradiator "Nordion"). There were 6 array holes in the ceiling and floor of the chamber through which the gamma irradiation passed. After irradiation, animals were placed on a warm pad or water bed until regained consciousness.

Histological examination. At selected time points, mice in each treatment group were euthanized and testis tissues examined were fixed in 10% formalin in PBS, pH 7.4 at 4 ° C for 24 hours. A small slit in the testicular capsule was created to allow the fixative to penetrate. Fixed samples were washed 4 times with PBS and embedded in paraffin using a tissue Tek-II tissue processor (MET). 8 μm
Sections were made, stained with hematoxylin and eosin (H & E), and coverslipped onto a glass slide on an Aquamount (Lerner Laboratories). The section was observed with a Zeiss or Olympic optical microscope with a 40x objective (
Total magnification 400x).

Quantitative histological analysis. Quantitative data were obtained from the testes of 2 animals in each treatment group 2 months after treatment (Table 1). In the control group, only one mouse was used. Single-section vas deferens were counted with a Zeiss optical microscope using a 5x objective (50x total magnification).
. Each vas deferens was examined at 400x total magnification. The seminiferous tubule has almost no cells in it,
The tube consisted of a single cell layer of basement membrane and was considered severely impaired when most of the spermatogonia along the basement membrane were present. The moderately injured tube had a part of the spermatogonia layer near the lumen peeled off. The spermatogonia heads were counted in the tubes and the average value was given by the total number of tubes counted.

Results of histological analysis. Up to 2 weeks after treatment, no obvious histological changes in testis were observed (data not shown). In FIG. 1, the time course of the testis of a mouse treated with the combination therapy of the alkylate agent busulfan and gamma irradiation as described above is shown.

FIG. 1A shows a 400 × image of seminiferous tubules obtained from a mouse 2 weeks after Busulfan 400 Rad treatment. In FIG. 1A, spermatogenesis was severely disrupted. All mature sperm disappeared and no spermatids or spermatocytes appeared. In the peripheral layer along the basement membrane, a small number of Sertoli cell nuclei and spermatogonia were found.

FIG. 1B shows a 400 × image of seminiferous tubules obtained from a mouse 6 weeks after Busulfan 400 Rad treatment. In FIG. 1B, a reconstructed image of spermatogenesis was seen. Not only spermatocytes but also sperm cells and sperm were seen.

After about 3 months, most seminiferous tubules were at least partially recovered and spermatogenesis at all stages appeared (data not shown). FIG. 1C shows a 400 × image of seminiferous tubules obtained from a mouse 5 months after treatment with Busulfan 400 Rad. At this stage, spermatogenesis had returned to normal.

Comparisons were also made in the three treatment groups mentioned above. The most dramatic difference between the groups was seen at 2 months post treatment. Two months after treatment, the group of mice receiving busulfan 400 Rad and γ-irradiation combination therapy showed the highest number of seminiferous tubules (Fig. 2).
(See B). The seminiferous tubules in this group contained fewer sperm heads per seminiferous tubule and severely and moderately damaged seminiferous tubules than the other treatment groups and control mice (see Table 1). Treatment with 400 Rad gamma irradiation and busulfan alone resulted in spermatozoa combined with cells in the lumen and mature sperm as compared to controls, to a significantly lesser extent than indicated by the Busulfan 400 Rad treatment group. There was damage during the formation process.

These results demonstrate that the combined therapy of alkylate and gamma irradiation is a more effective approach than destroying the testes, which were the tissues of male germ cells, in males than either treatment alone.

[Table 2]

Example 14 In Vivo Gene Transfer Using Lentiviral Vectors Lentiviral vectors were used in gene transfer into germ cells of male mice (usually for the purpose of transforming or modifying). In particular, instead of the LacZ promoter
Green Flourescent P under the transcriptional control of CMV (cytomegalovirus) promoter
A Carlos et al. modified pseudo-HIV (human AIDS virus) derived lentiviral vector (HR'GFP) was used to express rotein (GFP) (Naldini et al., Sc
ience 272: 263-67 [1996], "Gene transfer to non-dividing cells and stable gene transfer using lentiviral vectors").

Recipient C57BL / 6J mice were infected with the virus and treated with busulfan 44 days later. Male C57BL / 6J mice were injected with 0.1 ml of busulfan into the peritoneum at a concentration of 2 mg / ml. The dose was 4μ busulfan / gm body weight. Pretreated mice were anesthetized with avertin (0.017 mls / gm body weight per body weight) and the right testis was exposed after midline abdominal incision.

Lymphatic vessels were removed from adipose tissue and HIV-derived GFP vector (HR′GFP) was added to 1 × 10 5.
Particles were injected into the seminiferous tubules of the right testis from the lymphatic vessels of C57BL / 6J mice treated with 10 μl of busulfan at a particle / ml ratio. Injection was performed using a quartz glass micropipette equipped with Picospritzer II. The Picospritzer II was set at 80 psi and I pedaled for 1 second. The seminiferous tubules of the testis were all reached with a single injection so that the lymphatic vessels lead to a common chamber. As a control, the left testis was not injected.
Gene transfer into testicular cells in the duct was well spread.

Twenty-one days after treatment, the mice were killed and their testes were washed with 4% paraformaldehyde (pH7.4).
As a PBS solution) at 4 ° C. overnight. Wash the fixed testis 3 times with PBS solution,
Incubate overnight at 4 ° C in 20% sucrose. The testes were frozen by OCT and 8 μm thick sections were prepared using a cryostat. After storage at room temperature, it was washed with PBS solution and examined under a Zeiss 310 confocal microscope. The wavelength used for microscopy was 488 nm.

Green fluorescence was seen in all microscopic tubes examined, but the intensity was greatest in the tube on the surface of the testis. Gene transfer was observed in testis cells and Sertoli cells along the basement membrane (see FIG. 3), but almost never in spermatocytes or spermatids. Few sperms matured with busulfan treatment were found. No fluorescence was observed in the left testis used as a control. From these results, it was found that gene transfer using a lentivirus-derived vector was effective in male germ cells.

Although the above example is shown, it is not an exhaustive description of the realization of the present invention. The following description is made as the scope of claims.

[Brief description of drawings]

FIG. 1 shows an H & E-stained mouse (C57BL / 6J strain) testis section treated with gamma-ray irradiation in combination with busulfan (busulfan / 400 Rad treatment) (magnification 400 ×). FIG. 1A shows several sections of mouse seminiferous tubules 2 weeks after Busulfan / 400 Rad treatment. FIG. 1B shows several sections of mouse seminiferous tubules 6 weeks after Busulfan / 400 Rad treatment. FIG. 1C shows several sections of mouse seminiferous tubules 5 months after Busulfan / 400 Rad treatment.

FIG. 2 shows a histological comparison of three different methods and controls of depleting male germ cells from vertebrate testes. The H & E stained mouse (C57BL / 6J strain) section (magnification 400 times) collected 2 months after the treatment is shown. FIG. 2A shows a section of testis tissue after treatment with busulfan (4 mg / kg). FIG. 2B shows a section of testis tissue after the combined busulfan / 400 Rad treatment. FIG. 2C shows a section of testis tissue after treatment with 400 Rad gamma irradiation. FIG. 2D shows a section of testis tissue from an untreated control C57BL / 6J mouse 2 months after the start of the experiment.

FIG. 3 shows gene transfer to mouse testis cells in vivo using a lentiviral vector. Images were collected on a Zeiss 310 confocal light microscope (400x magnification). The HIV-based lentiviral vector contained a gene encoding GFP under the control of the CMV promoter. FIG. 3A shows transduced Sertoli cells, a maximum intensity projection of 19 images. FIG. 3B shows genetically modified (transduced) spermatogonia along the basal lamina of the seminiferous tubules.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 1/00 A61P 1/00 4C086 3/10 3/10 4C087 9/10 9/10 4C206 9/12 9 / 12 15/08 15/08 25/14 25/14 25/18 25/18 25/24 25/24 C12N 5/10 A61K 31/192 15/09 31/255 // A61K 31/192 31/573 31 / 255 31/675 31/573 35/52 31/675 35/76 35/52 C12N 15/00 A 35/76 5/00 B 38/00 A61K 37/02 (31) Priority claim number PCT / US99 / 08277 (32) Priority date April 15, 1999 (April 15, 1999) (33) Priority claiming country United States (US) (31) Priority claim number 09 / 292,723 (32) Priority date 1999 April 15, 1999 (April 15, 1999) (33) Priority claiming countries United States (US) (81) Designated countries EP (AT, BE, CH, CY, DE, DK, S, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD , GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, K, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Winston, Robert London England UK Denman Drive 11 F Term (reference) 4B024 AA01 CA02 DA02 DA03 EA02 FA02 HA01 HA17 4B065 AA90 AA93 AB04 AB06 AC14 BA02 CA43 CA44 4C076 AA19 AA95 BB11 BB16 BB32. 4C086 AA01 AA02 DA38 MA01 MA04 MA24 MA66 MA67 NA14 ZA12 ZA15 ZA18 ZA36 ZA42 ZA45 ZA66 ZA81 ZC35 4C087 AA01 AA02 BB60 BB65 CA04 MA24 MA66 MA67 NA13 MA04 NA04 MA04 NA04 MA04 NA04 MA04 NA04 NA04 ZA36 AZ81 ZA45 ZA45 ZA45 ZA45 ZA45 ZA45 ZA45 ZA12 ZA15 ZA18 ZA36 ZA42 ZA45 ZA66 ZA81 ZC35

Claims (159)

[Claims] 1. A method of substantially depleting germ cells from a vertebrate testis, comprising administering a dose of an alkylating agent to a male vertebrate and gamma-irradiating the male vertebrate with a gamma irradiation dose. The above method, which is used in combination in an amount sufficient to substantially deplete germ cells from the testis of the vertebrate. 2. The method of claim 1, wherein the alkylating agent is busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylethanesulfonic acid. 3. The dose of alkylating agent is about 4-10 mg / kg body weight.
The method described. 4. The method of claim 1, wherein the gamma irradiation is specifically directed to the testes of a male vertebrate and the gamma irradiation dose is about 200-800 Rads. 5. The method of claim 4, wherein the gamma irradiation dose is about 350-450 Rads. 6. A method of transfecting a substantially decellularized testis of a recipient male vertebrate with a maturing male germ cell genetically modified with at least one polynucleotide encoding a desired trait or product. Wherein at least one transfectant, at least one polynucleotide encoding a desired trait or product, and at least one gene encoding a gene selectable marker in the donor male vertebrate gonads A gene transfer mixture consisting of the polynucleotides of
Administration under conditions effective to reach at least one of the plurality of); the polynucleotide encoding the desired trait or product is taken up by and released into the germ cell (s) or precursor (s) thereof. At least one polynucleotide encoding a desired trait or product and at least one encoding a gene selectable marker from a donor male vertebrate
Isolating or selecting at least one male germ cell (s) in a genetically modified maturation process having one polynucleotide; Recipient male vertebrate is genetically modified reproduction from a donor male vertebrate Recipes of genetically modified germ cells (s) so isolated or selected, having at least one testis substantially depleted of male germ cells by the method of claim 1, prior to administration of the cells. Administering to at least one testis of a vertebrate male vertebrate; and allowing the administered germ cells to remain in the seminiferous tubules of the recipient male vertebrate. 7. The method of claim 6, wherein the donor vertebrate is the same as the recipient vertebrate. 8. The method of claim 6, further comprising the step of incorporating a polynucleotide encoding the desired trait or product into the germ cell genome. 9. The method of claim 6, wherein the genetically male germ cells consist of undifferentiated male germ cells. 10. Gene transfer is carried out under temperature conditions of about 25 ° C. to about 38 ° C.
The method of claim 6. 11. The transfecting substance is a liposome, a viral vector,
7. The method of claim 6, comprising a transferrin / polylysine-enhanced viral vector, a lentivirus vector, an adenovirus vector, a retroviral vector, or other uptake-enhancing DNA segment, or a mixture of any of these. 12. The transfecting substance comprises a lentivirus vector, a retrovirus vector, an adenovirus vector, a transferrin / polylysine-enhanced adenovirus vector, a human immunodeficiency virus vector, a Moloney murine leukemia virus-derived vector, a mumps vector, and a germ cell. Virus-derived DN that facilitates uptake and release of polynucleotides into the cytoplasm
12. The method according to claim 11, consisting of a viral vector selected from the group consisting of ases, or the transfecting substance consisting of a functional fragment or mixture thereof. 13. The method of claim 11, wherein the transfecting agent comprises an adenovirus-derived vector having endosomolytic activity and the polynucleotide is operably linked to the vector. 14. The method of claim 6, wherein the polynucleotide encoding the desired trait or product is complexed with a viral vector. 15. The method of claim 6, wherein the transfecting agent comprises a lipid transfecting agent. 16. The method of claim 6, wherein the transfecting agent further comprises at least one polynucleotide encoding a male germ cell targeting molecule or gene selectable marker. 17. A male germ cell targeting molecule directed specifically to spermatogonia and consisting of a c-kit ligand; and a gene selectable marker consisting of a gene encoding a detectable product, the expression of said gene , Essentially cyclin A1 promoter, c-kit promoter, B-Myb promoter, c-raf-1 promoter, ATM (ataxia-telangiectasia) promoter, RBM (ribosome binding motif) promoter, DAZ (in azoospermia) 17. A promoter selected from the group consisting of (deleted) promoter, XRCC-1 promoter, HSP 90 (heat shock gene) promoter, and FRMI (from fragile X site) promoter. Method. 18. The method of claim 6, wherein the vertebrate is a mammal. 19. The method of claim 18, wherein the mammal is a human. 20. The method of claim 18, wherein the mammal is a human, non-human primate, livestock, or marine mammal. 21. A polynucleotide encoding a desired trait or product comprises:
7. The method of claim 6, which is derived from a vertebrate of the same species as the recipient vertebrate. 22. The method of claim 6, wherein the polynucleotide encoding the desired trait or product is of human origin. 23. The method of claim 6, wherein the vertebrate is selected from the group consisting of wild and domestic vertebrates. 24. A polynucleotide encoding a desired trait or product is
Mammals selected from the group consisting of humans and non-human primates, dogs, mice, rats, gerbils, hamsters, rabbits, cats, pigs, domestic mammals, pachyderms, marine mammals, horses, sheep and cattle, or the essence Ducks, geese,
The method according to claim 6, which is derived from a bird selected from the group consisting of turkey, chicken, ostrich, emu, guinea fowl, pigeon, and quail. 25. A non-human transgenic vertebrate, which is a recipient male vertebrate of the method of claim 6 or a progeny thereof, which consists of an original germ cell having at least one heterologous polynucleotide in its genome. Transgenic vertebrates. 26. The transgenic non-human vertebrate of claim 25, wherein the polynucleotide comprises at least one biologically functional gene. 27. The transgenic non-human vertebrate of claim 26, which is male. 28. The transgenic vertebrate of claim 27, which has native male germ cells transfected with a heterologous polynucleotide. 29. A progeny obtained by mating a transgenic non-human vertebrate or a male progeny thereof according to claim 27 with a female animal of the same species. 30. At least one obtained by crossing a vertebrate or progeny thereof according to claim 25 with an opposite animal of the same species and selecting the crossed progeny for the presence or absence of the transfected polynucleotide. A non-human vertebrate having a heterologous polynucleotide sequence in its germ cell. 31. The non-human vertebrate of claim 30, selected from the group consisting of mammals and birds. 32. A polynucleotide encoding a desired trait or product is
Mammals selected from the group consisting of humans and non-human primates, dogs, mice, rats, gerbils, hamsters, rabbits, cats, pigs, domestic mammals, pachyderms, marine mammals, horses, sheep and cattle, or the essence Ducks, geese,
31. The non-human vertebrate of claim 30, derived from a bird selected from the group consisting of turkey, chicken, ostrich, emu, guinea fowl, pigeon, and quail. 33. The non-human vertebrate of claim 30, wherein the non-human vertebrate is a duck, goose, turkey, chicken, ostrich, emu, guinea fowl, pigeon, or quail. 34. The non-human vertebrate of claim 30, wherein the mammal is a domestic or marine mammal. 35. The non-human vertebrate of claim 32, wherein the mammal is a bull. 36. The non-human vertebrate of claim 32, wherein the mammal is porcine or ovine. 37. The method of claim 30, which is selected from the group consisting of wild and domestic animals.
The non-human vertebrate described. 38. A vertebrate-derived germ cell according to claim 31, which comprises a native germ cell having at least one heterologous polynucleotide in its genome. 39. A vertebrate sperm comprising the germ cell of claim 38. 40. The method of gene therapy according to claim 6, wherein the polynucleotide encoding the desired trait or product is derived from a vertebrate of the same species as the recipient vertebrate. 41. A transgenic non-human vertebrate produced by the method of claim 6, wherein the polynucleotide encoding the desired trait or product is derived from any genome. 42. A method of transfecting a substantially decellularized testis of a recipient male vertebrate with a maturing male germ cell genetically modified with at least one polynucleotide encoding a desired trait or product. Wherein at least one transfectant, at least one polynucleotide encoding a desired trait or product, and at least one gene encoding a gene selectable marker in the donor male vertebrate gonads A gene transfer mixture consisting of the polynucleotides of
Administration under conditions effective to reach at least one of the plurality of); the polynucleotide encoding the desired trait or product is taken up by and released into the germ cell (s) or precursor (s) thereof. At least one polynucleotide encoding a desired trait or product and at least one encoding a gene selectable marker from a donor male vertebrate
Isolating or selecting at least one male germ cell (s) in a genetically modified maturation process having one polynucleotide; Recipient male vertebrate is genetically modified reproduction from a donor male vertebrate Having at least one testis substantially depleted of male germ cells by combining a dose of alkylating agent and a dose of gamma irradiation prior to administration of the cells, thus isolated or selected Administering the modified genetically modified germ cell (s) to at least one testis of a recipient male vertebrate; and thereby transferring the genetically modified mature germ cells to the recipient. Staying in the seminiferous tubules of a male vertebrate; 43. The alkylating agent is busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylethanesulfonic acid.
The method described. 44. The dose of alkylating agent is about 4-10 mg / kg body weight.
The method described in 42. 45. The method of claim 42, wherein the gamma irradiation dose is specifically directed to the testes of a male vertebrate and the gamma irradiation dose is about 200-800 Rads. 46. The method of claim 45, wherein the gamma irradiation dose is about 350-450 Rads. 47. An in vitro method of incorporating a polynucleotide encoding a desired trait or product into a maturing male germ cell, wherein at least one mating male germ cell is obtained from a donor male vertebrate; In the presence of a gene transfer mixture comprising at least one transfectant, at least one polynucleotide encoding a desired trait or product, and at least one polynucleotide encoding a gene selectable marker Transfecting the germ cell (s) in vitro with at least one polynucleotide encoding the desired trait or product for a period effective at gene transfer at or below body temperature; Ingested by germ cells (plural) A genetically modified germ cell having at least one polynucleotide encoding a desired trait or product and at least one polynucleotide encoding a gene selection marker. Isolating or selecting at least one germ cell (s) from in vitro gene transfer with the aid of a gene selectable marker; the recipient male vertebrate is the genetically modified germ cell (s) isolated or selected. ) Is administered to a testis of a recipient male vertebrate having at least one testis substantially depleted of male germ cells not genetically modified by the method of claim 1, thus Administering the isolated or selected genetically modified germ cell (s); and administering the germ cell (s) administered. Said process comprising: step of stay in the seminiferous tubules of Piento male vertebrates. 48. The method of claim 47, further comprising the step of incorporating the released polynucleotide into the genome (s) of the genetically modified germ cell (s). 49. The method of claim 47, wherein the polynucleotide encoding the desired trait is integrated into the genome of a vertebrate germ cell. 50. The method of claim 47, wherein the maturing male germ cells comprise spermatogonia or other undifferentiated male germ cells. 51. The method of claim 47, wherein gene transfer is performed under temperature conditions of about 25 ° C to about 38 ° C. 52. The transfecting substance is a liposome, a viral vector,
48. The method of claim 47, which is a transferrin-polylysine-enhanced viral vector, a retrovirus, an adenovirus vector, a lentiviral vector, or other uptake-enhancing DNA segment, or a mixture of any of these. 53. The transfecting substance is a lentivirus vector, a retrovirus vector, an adenovirus vector, a transferrin / polylysine-enhanced adenovirus vector, a human immunodeficiency virus vector, a Moloney murine leukemia virus-derived vector, a mumps vector, or a germ cell. Virus-derived DN that facilitates uptake and release of polynucleotides into the cytoplasm
48. The method of claim 47, which comprises a viral vector selected from the group consisting of ases, or wherein the transfecting agent consists of a functional fragment or mixture thereof. 54. The method of claim 47, wherein the transfecting agent comprises an adenovirus vector having endosomolytic activity, and the polynucleotide encoding the desired trait is operably linked to the vector. 55. The method of claim 47, wherein the polynucleotide encoding the desired trait forms a complex with a viral vector. 56. The method of claim 47, wherein the transfectant comprises a lipid transfectant. 57. The method of claim 47, wherein the transfecting agent further comprises a male germ cell targeting molecule. 58. The male germ cell targeting molecule is specifically directed to spermatogonia and consists of a c-kit ligand; and the polynucleotide encoding a gene selectable marker consists essentially of the cyclin A1 promoter, c-kit. Promoter, B-Myb promoter, c-raf-1 promoter, ATM (ataxia-telangiectasia) promoter, RBM (ribosome binding motif) promoter, DAZ (deleted in azoospermia) promoter, XRCC-1 58. The gene of claim 57, which comprises a gene that expresses a detectable product promoted by a promoter selected from the group consisting of a promoter, the HSP 90 (heat shock gene) promoter, and the FRMI (from the fragile X site) promoter. Method. 59. The method of claim 47, wherein the vertebrate is a mammal. 60. The method of claim 59, wherein the mammal is a human. 61. The method of claim 59, wherein the mammal is selected from the group consisting of humans and non-human primates and domestic and marine mammals. 62. The method of claim 47, wherein the polynucleotide encoding the desired trait is derived from a vertebrate of the same species as the maturing germ cell. 63. The method of claim 47, wherein the vertebrate is selected from the group consisting of wild and domestic vertebrates. 64. A polynucleotide encoding a desired trait or product comprises:
Mammals selected from the group consisting of humans and non-human primates, dogs, mice, rats, gerbils, hamsters, rabbits, cats, pigs, domestic mammals, pachyderms, marine mammals, horses, sheep and cattle, or the essence Ducks, geese,
48. The method of claim 47, which is derived from a bird selected from the group consisting of turkey, chicken, ostrich, emu, guinea fowl, pigeon, and quail. 65. The method of claim 64, wherein the polynucleotide is of human origin. 66. A non-human transgenic comprising a native germ cell having at least one heterologous polynucleotide in its genome, wherein the polynucleotide has been integrated into the genome of the germ cell through the method of claim 47. Vertebrate or its offspring. 67. The alkylating agent is busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylethanesulfonic acid.
The method described. 68. The dose of alkylating agent is about 4-10 mg / kg body weight.
47. The method described. 69. Gamma irradiation is specifically directed to the testes of a male vertebrate,
48. The method of claim 47, wherein the gamma irradiation dose is about 200-800 Rads. 70. The method of claim 69, wherein the gamma irradiation dose is about 350-450 Rads. 71. The transgenic non-human vertebrate of claim 66, wherein the polynucleotide comprises at least one biologically functional gene. 72. The transgenic non-human vertebrate of claim 71, which is male. 73. The transgenic non-human vertebrate of claim 72, which has native male germ cells genetically modified by a heterologous polynucleotide. 74. A progeny obtained by mating a transgenic non-human vertebrate or a male progeny thereof according to claim 72 with a female animal of the same species. 75. At least one obtained by crossing the vertebrate of claim 66 or a progeny thereof with an opposite animal of the same species and selecting the progeny for the presence or absence of the transfected polynucleotide. A non-human vertebrate having in its germ cell a heterologous polynucleotide sequence. 76. The non-human vertebrate of claim 75, wherein the non-human vertebrate is selected from the group consisting of mammals and birds. 77. The mammal is a human or non-human primate, dog, mouse,
Selected from the group consisting of rats, gerbils, hamsters, rabbits, cats, pigs, domestic mammals, pachyderms, marine mammals, horses, sheep and cows, and the birds are essentially ducks, geese, turkeys, chickens, ostriches, 77. The method of claim 76, selected from the group consisting of emu, guinea fowl, pigeons, and quail. 78. The non-human vertebrate of claim 76, wherein the bird is a duck, goose, turkey, chicken, ostrich, emu, guinea fowl, pigeon, or quail. 79. The non-human vertebrate of claim 76, wherein the mammal is a domestic or marine mammal. 80. The non-human vertebrate of claim 76, wherein the mammal is a bull. 81. The non-human vertebrate of claim 76, wherein the mammal is pig or sheep. 82. The method of claim 75, wherein the group is selected from the group consisting of wild and domestic animals.
The non-human vertebrate described. 83. A germ cell obtained from the vertebrate of claim 66 or a progeny thereof. 84. A germ cell obtained from the vertebrate of claim 72. 85. A vertebrate sperm comprising a plurality of germ cells obtained from the vertebrate of claim 72. 86. A step of introducing a transfected male germ cell into a testis of a recipient vertebrate, wherein the polynucleotide encoding the desired trait or product is derived from a vertebrate of the same species as the recipient vertebrate. , Claims
47. The method described. 87. The transgenic non-human vertebrate produced by the method of claim 47, wherein the polynucleotide encoding the desired trait or product is derived from any genome. 88. From at least one transfectant or gene transporter, an alkylating agent, and optionally a gene selectable marker, whereby the kit is used to genetically modify and transfer germ cells in a viable state. A kit for the genetic modification and transfer of male vertebrate germ cells, which comprises the components of the gene transfer mixture comprising: 89. The alkylating agent is busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylethanesulfonic acid.
Kit described. 90. The kit of claim 88, further comprising a radiation shield capable of specifically directing gamma irradiation to the testis. 91. The transfecting or gene transporting agent is selected from the group consisting of liposomes, viral vectors, transferrin polylysine-enhanced viral vectors, retroviruses, lentiviral vectors, and uptake-enhancing DNA segments, or any of these. 89. The kit of claim 88, which consists of a mixture. 92. The transfecting or gene transporting substance is a lentivirus vector, retrovirus vector, adenovirus vector, transferrin / polylysine-enhanced adenovirus vector, human immunodeficiency virus vector, Moloney murine leukemia virus-derived vector, mumps vector, reproduction A viral vector selected from the group consisting of viral DNases that facilitates the uptake and release into the cytoplasm of cells of a cell, or the transfectant comprises a functional fragment or mixture of these, Claim 88
Kit described. 93. The kit of claim 88, wherein the transfecting or gene transporting agent comprises an adenovirus vector having endosomolytic activity, and the polynucleotide is operably linked to the vector. 94. The kit of claim 88, wherein the transfecting or gene transfer agent comprises a lipid transfecting agent. 95. The kit of claim 88, wherein the transfecting or gene transporting agent further comprises a male germ cell targeting molecule. 96. The kit of claim 95, wherein the male germ cell targeting molecule is specific for spermatogonial targeting and comprises a c-kit ligand. 97. The kit of claim 88, wherein the component comprises an immunosuppressant. 98. The kit of claim 97, wherein the immunosuppressive agent is cyclosporine or a corticosteroid. 99. A gene in which a male germ cell targeting molecule is specifically directed to spermatogonia and consists of a c-kit ligand; and a gene selectable marker expresses a detectable product driven by a spermatogonia specific promoter. 96. The kit of claim 95, which consists of: 100. A male germ cell targeting molecule is specifically directed to spermatogonia and consists of a c-kit ligand; and the gene selectable marker is a cyclin A1 promoter, c-kit promoter, BM.
yb promoter, c-raf-1 promoter, ATM (ataxia-telangiectasia) promoter, RBM (ribosome binding motif) promoter, DAZ (lacking in azoospermia) promoter, XRCC-1 promoter, HSP 90 96. The kit of claim 95, which comprises a gene that expresses a detectable product driven by a promoter selected from the group consisting of a (heat shock gene) promoter and a FRMI (from the fragile X site) promoter. 101. The kit of claim 88, wherein the at least one polynucleotide comprises a polynucleotide sequence encoding a gene selectable marker. 102. The kit of claim 101, wherein the encoded gene selectable marker is a fluorescent or light emitting protein. 103. The kit according to claim 102, wherein the fluorescent or light emitting protein is green fluorescent protein, yellow fluorescent protein, blue fluorescent protein, phycobiliprotein, luciferase, or aquaequorin. 104. An in vitro method for incorporating a polynucleotide encoding a desired trait or product into a maturing male germ cell, wherein at least one mating male germ cell is obtained from a donor male vertebrate; In the presence of a gene delivery mixture consisting of at least one transfecting agent, at least one polynucleotide encoding a desired trait or product, and at least one polynucleotide encoding a gene selection marker Transfecting the germ cell (s) in vitro with at least one polynucleotide encoding the desired trait or product for a period effective at gene transfer at or below body temperature; Uptake by germ cells Releasing into it, at least one polynucleotide, and at least one genetically modified germ cell having at least one polynucleotide encoding the gene selection marker, with the aid of the gene selection marker. Isolating or selecting from in vitro gene transfer, the recipient male vertebrate is prior to the administration of the isolated or selected germ cell (s), the genetically modified germ cell (s) from the donor male vertebrate. ) By administering a sufficient amount of alkylating agent dose and gamma irradiation dose prior to
Administering the genetically modified germ cell (s) thus isolated or selected to the testes of a recipient male vertebrate having at least one testis substantially depleted of endogenous male germ cells And retaining the administered germ cells in the seminiferous tubules of the recipient male vertebrate. 105. The alkylating agent is busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylethanesulfonic acid.
The method described in 104. 106. The method of claim 104, wherein the gamma irradiation dose is specifically directed to the testes of the male vertebrate and the gamma irradiation dose is about 200-800 Rads. 107. The gamma ray dose of about 350-450 Rads.
The method described. 108. The method of claim 6, wherein the polynucleotide encoding the desired trait or product is operably linked to a germ cell-specific promoter. 109. The method of claim 42, wherein the polynucleotide encoding the gene selectable marker is operably linked to a germ cell specific promoter. 110. The method of claim 47, wherein the polynucleotide encoding the gene selectable marker is operably linked to a germ cell specific promoter. 111. The method of claim 104, wherein the polynucleotide encoding the gene selectable marker is operably linked to a germ cell specific promoter. 112. The method of claim 6, wherein the feeder cells are co-administered to the testes with the isolated or selected germ cells. 113. The method of claim 42, wherein the feeder cells are co-administered to the testes with the isolated or selected germ cells. 114. The method of claim 47, wherein the transfected feeder cells are isolated or selected and are co-administered with the isolated or selected germ cells into the testes of the recipient male vertebrate. 115. The method of claim 104, wherein the genetically modified feeder cells are isolated or selected and are co-administered with the isolated or selected germ cells into the testes of the recipient male vertebrate. 116. The method according to claim 6, wherein the gene selection marker is a fluorescent protein or a light emitting protein. 117. The method according to claim 116, wherein the fluorescent or light emitting protein is green fluorescent protein, yellow fluorescent protein, blue fluorescent protein, phycobiliprotein, luciferase, or aquaequorin. 118. The method of claim 42, wherein the gene selectable marker is a fluorescent protein or a light emitting protein. 119. The method of claim 118, wherein the fluorescent or light emitting protein is green fluorescent protein, yellow fluorescent protein, blue fluorescent protein, phycobiliprotein, luciferase, or aquaequorin. 120. The method of claim 47, wherein the gene selectable marker is a fluorescent protein or a light emitting protein. 121. The method of claim 120, wherein the fluorescent or light emitting protein is green fluorescent protein, yellow fluorescent protein, blue fluorescent protein, phycobiliprotein, luciferase, or aquaequorin. 122. The method of claim 104, wherein the gene selectable marker is a fluorescent protein or a light emitting protein. 123. The method of claim 122, wherein the fluorescent or light emitting protein is green fluorescent protein, yellow fluorescent protein, blue fluorescent protein, phycobiliprotein, luciferase, or aquaequorin. 124. An in vivo method for incorporating a polynucleotide into a male vertebrate germ cell comprising at least one polynucleotide encoding a desired trait or product, and at least one lentivirus-derived transfecting or gene transporter. , And optionally, a gene transfer mixture consisting of a gene selectable marker into the testes of a male vertebrate under conditions effective to reach vertebrate germ cells or precursors thereof; and to reproduce the desired trait or product. The step of incorporating into cells or their precursors and releasing them thereinto; 125. The method of claim 124, further comprising the step of incorporating the released polynucleotide into the germ cell genome. 126. The method of claim 124, wherein the transfecting or gene transporting agent further comprises a male germ cell targeting molecule. 127. The method of claim 126, wherein the male germ cell targeting molecule is specific for spermatogonia targeting and is a c-kit ligand. 128. The method of claim 124, wherein the gene transfer mixture further comprises an immunosuppressant agent. 129. The method of claim 128, wherein the immunosuppressive agent is selected from the group consisting of cyclosporine and corticosteroids and the agent is administered systemically. 130. The gene transfer mixture is administered by injection.
4 Method described. 131. The method of claim 130, wherein the injection comprises subcutaneous injection of a vertebrate testis. 132. The method of claim 124, wherein the gene transfer mixture is administered directly to the vas deferens of vertebrates. 133. The method of claim 124, wherein the gene transfer mixture is administered directly to the vas deferens of the vertebrate testis. 134. The gene transfer mixture is administered directly to the vertebrate testicular net.
The method of claim 124. 135. The method of claim 124, wherein the vertebrate is a mammal. 136. The method of claim 135, wherein the mammal is a human. 137. The group wherein the mammal comprises humans and non-human primates, dogs, mice, rats, gerbils, hamsters, rabbits, cats, pigs, livestock mammals, pachyderms, marine mammals, horses, sheep and cows. 138. The method of claim 136, wherein the bird is selected from the group consisting of duck, goose, turkey, chicken, ostrich, emu, guinea fowl, pigeon, and quail. 138. The method of claim 124, wherein the vertebrate is selected from the group consisting of wild and domestic vertebrates. 139. The method of claim 124, wherein the polynucleotide encoding the desired trait or product is from the same species as the male vertebrate. 140. A transgenic non-human vertebrate or progeny thereof produced by the method of claim 124, wherein the polynucleotide encoding the desired trait or product is derived from any genome. 141. The transgenic non-human vertebrate animal of claim 140, which consists of native germ cells having at least one heterologous polynucleotide in its genome. 142. The transgenic non-human vertebrate of claim 141, wherein the polynucleotide comprises at least one biologically active gene. 143. The non-human transgenic vertebrate of claim 140, which is male. 144. A progeny obtained by mating the transgenic non-human vertebrate or the progeny thereof according to claim 143 with a female animal of the same species. 145. A non-human vertebrate crosses the vertebrate of claim 140 or a progeny thereof with an opposite animal of the same species, and selects progeny for the presence or absence of the transfected polynucleotide. A non-human vertebrate having in its germ cell at least one heterologous polynucleotide sequence obtained thereby. 146. The vertebrate other than humans is a mammal or a bird.
145. Non-human vertebrates according to 145. 147. The group wherein the mammal comprises humans and non-human primates, dogs, mice, rats, gerbils, hamsters, rabbits, cats, pigs, livestock mammals, pachyderms, marine mammals, horses, sheep and cows. 157. The non-human vertebrate of claim 146, selected from the group consisting of: duck, goose, turkey, chicken, ostrich, emu, guinea fowl, pigeon, and quail. 148. The vertebrate of claim 146, wherein the mammal is selected from the group consisting of wild and domestic mammals. 149. The vertebrate of claim 146, wherein the mammal is a domestic or marine mammal. 150. The vertebrate of claim 146, wherein the mammal is a bull, pig or sheep and the bird is a chicken. 151. A germ cell obtained from the vertebrate of claim 140. 152. Further, a step of producing a male vertebrate containing germ cell (s) after incorporating a polynucleotide encoding a desired trait or product into germ cells or their precursors and releasing them therein. And a step of recovering male germ cells produced by the male vertebrate, the vertebrate male germ cells obtained by a method comprising the method of claim 124; 153. The vertebrate of claim 152, wherein the method of obtaining germ cells further comprises the step of mating male vertebrates to produce progeny, and collecting the germ cells produced by the male progeny. Male germ cells. 154. A vertebrate sperm comprising the germ cell of claim 152. 155. A vertebrate sperm comprising the germ cell of claim 153. 156. A method of producing a non-human vertebrate strain consisting of native germ cells having at least one heterologous polynucleotide in its genome, wherein the vertebrate of claim 142 is the same species. The method described above, which comprises mating with an animal of opposite sex and selecting progeny for the presence of the polynucleotide. 157. The method of claim 157, wherein the gene transfer mixture comprises at least one polynucleotide encoding a gene selectable marker, and the genetically modified male germ cells are isolated or selected with the aid of the gene selectable marker. 124. A method for isolating or selecting male germ cells transfected with at least one polynucleotide encoding a desired trait or product and at least one gene selection marker, which comprises the method according to 124. 158. The method of claim 157, wherein the gene selectable marker is a fluorescent protein or a light emitting protein. 159. The method of claim 158, wherein the fluorescent or light emitting protein is green fluorescent protein, yellow fluorescent protein, blue fluorescent protein, phycobiliprotein, luciferase, or aquaequorin.