CA2183553A1 - Enhanced transgene expression in specific tissues of the gastrointestinal tract - Google Patents

Abstract

This invention provides a mammal with enhanced expression of a transgene in certain tissues of the gastrointestinal tract. Also provided are 1) a nucleic acid sequence useful in enhancing expression of a transgene in certain gastrointestinal tissues, and 2) a vector containing this nucleic acid sequence.

Description

~ WO95/11299 ~l ~ 35~3 PCr/US94/11716 ENHANCED TRANSGENE EXPRESSION IN SPECIFIC
GA~ lN~ IlNAL TRACT TISSUES OF THE
R~R~Rou~n 5 Field of the Inv, ntion This invention relates to the field of recombinant DNA technology, especially to nucleic acid sequences useful for constructlng a transgenic mammal.
10 More spe~ rz- l l y, the invention concerns expression of a transgene in certain tissues or organs of a non-human mammal .
Descriotion of Related ~rt Production of a transgenic mammal involves the insertion of a nucleic acid sequence, often called a transgene, which codes for a particular polypeptide, into one or more chromosomes of the mammal. This is 20 typically accomplished by inserting the transgene into the pronucleus of an isolated mammalian egg. The transgene becomes incorporated into the DNA of the developing embryo. This embryo is then implanted into a surrogate host for the duration of gestation. The 25 offspring of the surrogate host are evaluated for the presence of the transgene.
Expression of the transgene, ~.e., production of the protein encoded by the transgene nucleic acld sequence, may confer a new phenotype on the mammal.
30 Depending on the transgene (s) inserted into the animal and the level of expression of the transgene in the mammal, the mammal may become more or less susceptible to a particular disease or series of diseases. Such transgenic mammals are valuable for ~n vlvo screening 35 and testing of compounds that may be useful in treating 218~3 .
WO 95111299 ' PCTiUS9~/ll7l6 or preventing the disease (s), and/or for developing methods useful in dlagnosing the disease.
While methods for insertion of a novel gene into a mammal have been developed rapidly, several problems with the application of this technology remain.
One such problem concerns limiting expression of the gene prlmarily to a selected tissue or tissues where expression is desired.
Enhanced expression of some genes and transgenes in certain cells or tissues types appears to be directly regulated, at least in part, by the promoter. One such promoter is the intestinal fatty acid binding protein (FABP) promoter. ~his promoter, containing about 1.2 kb of 5 ' flanking sequence, has been demonstrated to confer lineage speciflc expression of certain transgenes in the gastro-intestinal villus enterocytes of mice. The transgenes evaluated include, for example, human growth hormone and SV40 ~-antigen (Rottman et al ., .T. 13iol. Chem., 268 :11994-12002 [1993];
Hauft et al., Clin. Res., 39:325A [1991]; Roth et al., Proc. Natl. Acad. Scf USA, 88:9407-9411 [1991]; Roth et al., Amer. J. Physiol., 63:GI86-G197 [1992]; Cohn et al., J. Cell. Biol., 119:27-44 [1992]; Kim et al., Surg.
~orum, 93 :153-155 rl~92] ) . Another promoter, the ApoC-III promoter, has been shown to confer enhanced gastro-intestinal expression of the human ApoC-III and ApoA-I
genes in transgenic mice (Walsh et al., J. Lipid Res., 34:617-623 [1993]). Similarly, a truncated liver fatty acid binding protein promoter has been shown to confer liver, kidney, and colonic crypt expression of human growth hormone in transgenic mice ~Roth et al, J. Biol.
Chem., 266:5949-5954 [1991]) .
The interleukins are a group of ~ naturally occurring proteins that act as chemical mediators of the differentiation processes for red and white blood cells.
One o~ the interleukins, IL-8 (also known as Neutrophil 2183~3 WO 95/11299 PcrluS94/11716 Activating Peptide-1, or NAP-1), has been shown to be a neutrophil chemoattractant with the ability to actlvate neutrophils and stlmulate the respiratory burst (Colditz et al., J. ~eukocyte ~ol., 48:129-137 [1990]; Leonard et al., J. Invest. Derm., 96: 690-694 [1991] ) . IL-8 has been termed a proinflammatory cytokine due to its involvement in neutrophil recruitment to sites of acute and chronic in~lammation.
Zwahlen et al. (Int. Rev. Exp. Path., 34B:22-42 [1993] ) describe some effects of IL-8 in~ected into some rodents. When in~ected intr~ rm l ly into rats, IL-8 induced neutrophil infiltration at the site of injection. Intravenous in~ection of IL-8 into rabbits resulted in neutrophil seSIuestration in the lungs.
Vogels et al. ~Antimicrob~al Agents and Chemotherapy, 37:276-280 [1993]) describe the effect of administering IL-8 to mice either before or after infection of the mice with three different pathogens.
Under certain conditions, administration of IL-8 was shown to have a detrimental effect on the survival of the mice.
Van Zee et al. (J. Immunol., 148:1746-1752 [1992] ) describe administration of IL-8 to baboons . The animals developed neutropenia rapidly after IL-8 administration. This neutropenia is transient and is followed by a marked granulocytosis which persists for as long as IL-8 is present in the circulation.
Burrows et al. (Ann. NY Acad. Sc~., 629:422-424 [1991] ) show that guinea pigs in~ected with IL-8 had a higher level of T-lymphocyte and eosinophil ~t~on in the lung than did control animals.
Keratinocyte growth factor (KGF) is a mitogen that has been identified as specific for epithelial cells, especially keratinocytes (Rubin et al., Proc.
Natl. Acad. Sci. USA, 86:802-806 [1989]; Finch et al., Science, 245:752-755 [1990]; Marchese et al., J. Cell 2~83~53 Wo 95/11299 ~ PCT/IIS94/11716 .,, Physiol., 144:326-332 [1990]) . KGF has shown potential for repair of epidermal tissues such as the skin, and epithelial tissues of the digestive tract. The DNA
encoding KGF has been cloned and sequenced (PCT
90/08771, p~lished August 9, l990).
Guo et al. (EMBO J., 12:973-986 [1993]) have prepared a transgenlc mouse containing a transgene constructed of the human keratin 14 promoter and the human keratinocyte growth factor gene. The mouse showed a number of phenotypic differences as compared with non-transgenics such as wrinkled skin and reduced hair follicle density.
Monocyte chemoattractant protein (also known as MCP-1) is a protein that is produced by activated leukocytes in response to certain stimuli. The gene encoding human MCP-l has been cloned and sequenced (Furutani et al., Biochem. B~ophys. Res. Comm., 159:249-255 [1989]; Yoshimura et al., Chemotactic Cyto~c~nes, Westwood et al., eds. Plenum Press, NY [1991], pp.47-56) . MCP-l serves to attract monocytes to the site of its release, and is believed to be involved in the cellular immune response and in acute tissue in~ury (Leonard et al., Immunol. Today, 11:97-101 [1990]).
MCP-l has been shown to be produced by some tumor cells in vitro, and in human metastatic melanomas ln vivo (Graves et al., Am J. Pathol., 140:9-14 [1992]) .
While many genes that appear to be important in a variety of diseases have been studied in ~n v~tro systems, there is a need in the art to provide ~n v~ vo systems to more accurately evaluate the role of these genes in disease.
:~ncnr~ ngly, it is an ob~ect of this invention to provide a mammal containing a nucleic acid construct encoding a transgene, wherein the transgene is expressed primarily in gastro-intestinal tissues.

WO 95/11299 ~ 1 8 3 $~ ~ PCT/US94/~ 1716 It is a further ob~ect to provide a nucleic acid construct and an expression vector that enhance tissue specific expression of a transgene in some gastrointestinal tissues.
Other such objects will readily be apparent to one o~ ordinary skill in the art.
SVMMARY OF THE INVENTION
In one aspect, the present invention provides a nucleic acid sequence comprising a transgene excluding human growth hormone, beta-galactosidase, and SV40 T
antigen, wherein the transgene is operably linked to a promoter selected from the group consisting of:
intestinal FA;3P promoter, liver FABP promoter, and ApoC-III promoter.
In another aspect, the invention provides a nucleic acid sequence comprising a transgene ~ lfl;ng human growth hormone, beta-galactosidase, and SV40 T
antigen, wherein the transgene is operably linked to a promoter selected from the group consisting of:
intestinal FABP promoter, liver FABP promoter, and ApoC-III promoter, and wherein the transgene is selected from the group of transgenes consisting of: interleukin 1, int~rl~llkin 2, interleukin 3, interleukin 4, interleukin 5, interleukin 6, interleukin 7, interleukin 8, interleukin 9, interleukin 10, interleukin 11, interleukin 12, ENA-78, interferon-cc interferon-p, interferon-y~ granulocyte-colony s~ t;ng factor, granulocyte-macrophage colony stimulating factor, macrophage colony stimulating factor, stem cell factor, keratinocyte growth factor, MCP-1 and TNF, and fragments thereof .

~1835~3 In still another aspect, the invention provides a nucleic acid sequence comprising the rat intestinal FABP promoter, the human ApoE intron l linked at its 5 ' end to the 3 ' portion of human ApoE exon l and 5 at its 3 ' end to the 5 ' portion of the human ApoE exon 2 and the coding sequence of the transgene human IL-8 or human XGF.
In one other aspect, the invention provides a l0 mammal or its progeny containing a nucleic acid sequence comprising at least a portion of transgene ~ r~ ; ng human growth hormone, beta-galactosidase, and SV 40 T
antigen, operably linked to a promoter selected from the group consi~ting of: liver f~tty acid bind protein 15 promoter, intestinal fatty acid binding protein promoter, and ApoC--III. ~
In yet another aspect, the invention provides a mammal wherein the nucleic acid sequence comprises the 20 rat intestinal FABP promoter, the human ApoE intron l linked at its 5 ' end to the 3 ' portion of the human ApoE
exon l and at its 3 ' end to the 5 ' portion of the human ApoE exon 2, and the transgene human ~L-8 or KGF.
In still one other aspect, the invention provides a eukaryotic cell r~ntA1ning a nucleic acid sequence set forth above.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure l depicts the rat fatty acid binding protein promoter (FABPp) sequence obtained from the full length FABP sequence as it appears in Genbank (access~rm number Ml8080).

WO 95/11299 Z l ~ 3 ~ $ 3 PCT/[IS94/11716 i Figure 2A-C depicts the overall clonlng strategy for preparatlon of the constructs used to make the IL-8 and the KGF transgenic mlce. "FABP-p" refers to the rat fatty acld blnding protein promoter sequence;
5 "ApoE*" refers a nuclelc acid sequence containing the human ApoE DNA sequence encoding the 3 ' portion of exon l, the entire sequence of intron l, and the 5 ' portion of exon 2; "SV40PA" refers to the SV40 polyadenylation sequence .
Flgure 3 deplcts the level of IL-8 and the level of circulatlng neutrophlls ln both control and transgenlc mlce. Flgure 3A shows serum IL-8 levels.
Figure 3B shows circulatlng neutrophil levels. NT
15 represents non-transgenic (control) mlce. The numbers refer to lndlvidual llnes of transgenlc mice used in the analysls .
DETAILED DESCRIPTION OF THE INVENTION
Defln~tions The term "operably llnked" refers to the arrangement of various nuclelc acld sequence elements 25 relatlve to each such that the elements are functionally connected and are able to interact with each other.
Such elements may include, without limitation, a promoter, an enhancer, a polyadenylation sequence, one or more introns and/or exons, and a coding sequence of a 30 gene of interest to be expressed ~i.e., the transgene).
The nucleic acid sequence elements, when properly oriented or operably linked, act together to modulate the activity of one another, and ultimately may af fect the level of exprëssion of the transgene. By modulate 35 is meant increasing, decreasing, or maintaining the level of activity of a particular element. The position 2~83~i~3 WO 95111299 ~! PCrlUS94/11716 of each element relative to other elements may be expressed in terms of the 5 ' terminus and the 3 ' terminus of each element, and the distance between any particular elements may be re~erenced by the number of 5 intervening nucleotides, or base pairs, between the elements .
The term "transgene" refers to a particular nucleic acid sequence encoding a polypeptide or a portion of a polypeptide to be expressed in a cell into lO which the nucleic acid sequence is inserted. ~he term "tr~nsgene " is meant to include ~ l ) a nucleic acid sequence that is not naturally found in the cell (i.e., a heterologous nucleic acid sequence); (2) a nucleic acid sequence that is a mutant form of a nucleic acid 15 sequence naturally found in the cell into which it has been inserted; (3) a nucleic acid sequence that serves to add additional copies of the same ( i e ., homologous ) or a similar nucleic acid sequence naturally occurring in the cell into which it has been inserted; or (4) a 20 silent naturally occurring or homologous nucleic acid sequence whose expression is induced in the cell into which it has been inserted. By '1mutant form" is meant a nucleic acid sequence that contains one or more nucleotides that are different from the wild-type or 25 naturally occurring sequence, i . e ., the mutant nucleic acid sequence contains one or more nucleotide substitutions, flP1~ n~, and/or insertions. In some cases, the transgene may also include a sequence encoding a leader peptide or signal sequence such that 30 the transgene product will be secreted from the cell.
The term "promoter" refers to a nucleic acid sequence that regulates, either directly or indirectly, the transcription of a corresponding nucleic acid coding sequence to which it is operably linked. The promoter 35 may function alone to regulate transcription, or, in some cases, may act in concert with one or more other , WO 95/11299 2 ~ 8 ~ 5 ~ 3 PCTIUS94/11716 _ g _ regulatory sequences such as an enhancer or silencer to regulate transcription of the transgene.
The term "rodent" refers to all members of the phylogenetic order Rodent~a, such as, for example, 5 mouse, rat, hamster, s~uirrel, or beaver.
The term "progeny" refers tQ all offspring of the transgenic mammal, and includes every g,~n~r~ n subse~uent to the originally transformed transgenic mammal .
Prep~ration of t~e Invention 1. Preparation of DN~ Constructs A. Selection of Tr~ncaene This invention contemplates expression of one or more transgenes primarily in certain of the gastro-intestinal tissue of a transgenic mammal. Included 20 within the scope of this invention is any transgene encoding a polypeptide to be expressed in intestinal tissue. Typically, the transgene will be a nucleic acid seauence encoding a polypeptide involved in the immune response, inflammation, cell growth and proliferation, 25 cell lineage differentiation, and/or the stress response. The tr~nsgene may be homologous or heterologous to the promoter and/or to the mammal. In addition, the transgene may be a full length cDNA or genomic DNA sequence, or any fragment, subunit or mutant 30 thereof that has at least some biological activity.
Optionally, the transgene may be a hybrid nucleic acid sequence, ~.e., one constructed from homologous and/or heterologous cDNA and/or genomic DNA fragments. The transgene may also optionally be a mutant of one or more 35 naturally occurring cDNA and/or genomic sequences.

2~83~3 WO95/11299 PCTn7S94/11716 . ~ ! i The transgene may be isolated and obtained in suitable quantity using one or more methods that are well known in the art. These methods and others useful for isolating a transgene are set forth, for example, in 5 Sambrook et al. (M~7~c~7Ar Clonlng: A Laboratory Nanual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [1989] ) and in Berger and Kimmel (Methods in Enzymology: Gu~de to Molecular Cloning Technlques, vol. 152, Academic Press, Inc., San Diego, CA [1987] ) .
Where the nucleic acid sequence of the transgene is known, the transgene may be synthesized, in whole or in part, using chemical synthesis methods such as those described in Engels et al. (Angew. Chem. ~nt.
l~d. ~ngl., 28:716-734 [1989]). These methods include, inter alia, the phosphotriester, phosphoramidite and H-phosphonate methods of nucleic acid synthesis.
Alternatively, the transgene may be obtained by screening an appropriate cDNA or genomic library using one or more nucleic acid probes (oligonucleotides, cDNA or genomic DNA fragments with an acceptable level of homology to the transgene to be cloned, and the like) that will hybridize selectively with the tran3gene DNA.
Another suitable method for obtaining a transgene is the polymerase chain reaction (PCR).
However, successful use of this method requires that enough information about the nucleic acid sequence of the transgene is known in order to design suitable oligonucleotide primers useful for amplification of the appropriate nucleic acid sequence.
h7here the method of choice requires the use of oligonucleotide primers or probes ( e . g. PCR, cDNA or genomic library screening), the oligonucleotide sequences selected as probes or primers should be of adequate length and sufficiently unambiguous so as to minimize the amount of non-specific binding that will occur during library screening or PCR. The actual WO95111~99 2 ~ 3 PCT/US94/1~71C
sequence of the probes or prlmers is usually based on conserved or highly homologous sequences or regions from the same or a similar gene from another organism.
Optionally, the probes or primers can be degenerate.
In cases where only the amino acid sequence of the transgene is known, a probable and functlonal nucleic acid sequence may be inferred for the transgene using known and preferred codons for each amino acid residue. This sequence can then be chemically synth~s~ 7ed .
This invention contemplates the use of transgene mutant sequences. A mutant transgene is a transgene containing one or more nucleotide substitutions, deletions, and/or insertions as compared to the wild type sequence. The nucleotide substitution, deletion, and/or insertion can give rise to a gene product (i.e., protein) that is different in its amino acid sequence from the wild type amino acid sequence.
Preparation of such mutants is well known in the art, and is described for example in Wells et al. (Gene, 34:315 [1985]), and in Sambrook et al, supra.
Preferred transgenes of the present invention include erythropoietin (EPO), interleukin 1 (I~.-1), interleukin 2, interleukin 3, interleukin 4, interleukin 5, interleukin 6, interleukin 7, interleukin 8, interleukin 9, interleukin 10, interleukin 11, interleukin 12, ENA-78 (Walz et al., J. Exp. Med., 174:1355-1362 [1991]; Strieter et al., Immunol. Invest., 21:589-596 [1992]), interferon-,interferon-~, interferon-~y, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), stem cell factor (SCF), keratinocyte growth factor (RGF), monocyte chemoattractant protein-1 (MCP-1;
Furutani et al., supra), tumor necrosis factor (TNF), and fragments, subunits or mutants thereof. More 2183~5~
. . . .
WO95/11299 PCr/US94/11716 preferred transgenes include erythropoietin, interleukin 8, MCP-l, keratinocyte growth factor, and ENA-78. The most preferred transgenes include human interleukin 8, human keratinocyte growth factor, and MCP-l.

B . Selection of Regul ~torv El^--^nts This invention contemplates the use of promoters that enhance transgenic expression ln some lO gastro-intestinal tissues. These promoters may be homologous or heterologous to the transgene and/or to the transgenic mammal. Thus, the promoters used to pr2ctice this invention may be obtained from any source.
Preferred promoters of this group include the intestinal 15 fatty acid binding protein promoter (FABP promoter), the liver FABP promoter, and the ApoC-III promoter. The most preferred promoter of ~his group is the rat intestinal FABP promoter.
The promoter sequences of this invention may 20 be obtained by any of several methods well known in the art. Typically, promoters useful herein will have been previously identified by mapping and/or by restriction endonuclease digestion and can thus be isolated from the proper tissue source using the appropriate restriction 25 endonucleases. In some cases, the promoter may have been sequenced. For those promoters whose DNA sequence is known, the promoter may be synth~s~ 7~d using the methods described above for transgene synthesis.
Where all or only portions of the promoter 30 sequence are known, the promoter may be obtained using PCR and/or by screening a genomic library with suitable oligonucleotide and/or promoter sequence fragments from the same or another species.
Where the promoter sequence is not known, a 35 fragment of DNA containing the promoter may be isolated from a larger piece of DNA that may contain, for ~ WO95/11299 2~8~553 PCT/US94/~1716 example, a coding sequence or even another gene or genes. Isolation may be accomplished by restriction ~n~ nl1~1e~qe digestion using one or more carefully selected enzymes to isolate the proper DNA f ragment .
5 After digestion, the desired fragment is isolated by agarose gel purification, Qiagen column or other methods known to the skilled artisan . Sel ect i ~n of suitable enzymes to accomplish this purpose will be readily apparent to one of ordinary skill in the art.
C. Selection of Other Vector Cl on~ntS
In addition to the transgene and the promoter, the vectors useful in this invention typically contain 15 one or more other elements useful for ~l) optimal functioning of the vector in the mammal into which the vector is transfected, and ~2) amplification of the vector in bacterial or 1; ~n host cells . Each of these e1 q will be positioned appropriately in the 20 vector with respect to each other element so as to ; 7e their respective activities. Such positioning is well known to the ordinary skilled artisan. The following elements may be optionally included in the vector as appropriate.
i. Signal Se~auence El~m~nt For tho~e embodiments of the invention where the transgene is to be secreted, a signal 3equence, i5 30 frequently present to direct the polypeptide encoded by the transgene out of the cell where it is synthesized.
Typically, the signal sequence is positioned in the coding region of the transgene towards or at the 5 ' end of the coding region. Many signal sequences have been 35 identified, and any of them that are ~lln~-t~7n~1 in the transgenic tissue may be used in con~unction with the ~183Cj~3 transgene. Therefore, the signal seauence may be homologous or heterologous to the transgene, and may be homologous or heterologous to the transgenic mammal Additionally, the signal sequence may be chemically 5 synthesized using methods set forth above. However, for purposes herein, preferred signal sequences are those that occur naturally with the transgene (i.e., homologous to the transgene) .
ii. 1- rane ~nchor;nq r ln Element In some cases, it may be desirable to have a transgene expressed on the surface of a particular intr~c~ r membrane or on the plasma membrane.
15 Naturally occurring r~n~ proteins contain, as part of the translated polypeptide, a stretch of amino acids that serve to anchor the protein to the membrane.
However, for proteins that are not naturally found on the membrane, such a stretch of amino acids may be added 20 to confer this feature. Freq!uently, the anchor domain will be an internal portion of the protein and thus will be engineered internally into the transgene. However, in other cases, the anchor region may be attached to the 5' or 3' end of the transgene. Here, the anchor domain 25 may first be placed into the vector in the appropriate position as a separate component from the transgene. As for the signal seauence, the anchor domain may be from any source and thus may be homologous or heterologous with respect to both the transgene and the transgenic 30 mammal. Alternatively, the anchor domain may be chemically synthesized using methods set forth above.
iii. Oria,l n of Replication El,om~nt This ~ one.~t is typically a part of prokaryotic expression vectors purchased commercially, ~8~3 Wo 95/11299 PCT/US94/11716 and aids ln the amplification of the vector in a host cell. I the vector of choice does not contain an origin of replication site, one may be chemically synth~s; 7~'~ based on a known sequence, and ligated into 5 the vector.
iv. Tr~n~cri~tion T~rm; n~tion El ement This element is typically located 3 ' to the lO transgene coding sequence and serves to terminate transcription of the transgene. Usually, the transcription termination element is a polyadenylation signal sequence. While the element is easily cloned from a library or even purchased commercially as part of 15 a vector, it can also be readily synthesized using methods for nucleic acid synthesis such as those described above.
v. Tntron El~ ~nt In many cases, transcription of the transgene is increased by the presence of one or more introns on the vector. The intron may be naturally occurring within the transgene se~uence, especially where the 25 transgene is a full length or a fragment of a genomic DNA sequence. Where the intron is not naturally occurring within the DNA sequence (as for most cDNAs), the intron (s) may be obtained from another source. The intron may be homologous or heterologous to the 30 transgene and/or to the transgenic mammal. The position of the intron with respect to the promoter and the transgene is important, as the intron must be transcribed to be effective. As such, where the transgene is a cDNA sequence, the preferred position for 35 the intron is 3 ' to the transcription start site, and 5 ' to the polyA transcription termination sequence.

WO 95/11~99 PCT/US94/11716 Preferably for cDNA transgenes, the lntron will be located on one side or the other (i.e., 5' or 3') of the transgene sequence such that it does not interrupt the transgene sequence. Any intron from any source, 5 including any viral, prokaryotic and eukaryotic (plant or animal) organisms, may be used to practice this invention, provided that it is compatible with the host cell (s) into which it is inserted. Also included herein are synthetic introns. Optionally, more than one intron l0 may be used in the vector. A preferred intron is intron l of the human ApoE gene.
vi. Selectable I~Arker(s) Rl~ t Selectable marker genes encode proteins necessary for the survival and growth of transfected cells grown in a selective culture medium. Typical s~l~c~ n marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, e . g., ampicillin, tetracycline, or kanomycin for prokaryotic host cells, and neomycin, hygromycin, or methotrexate for mammalian cells; (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for cultures of Baclll ~ .
All of the elements set forth above, as well as others useful in this invention, are well known to the skilled artisan and are described, for example, in Sambrook et al. (M~7ec~7Ar Clon~ng:A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring ~arbor~
NY [1989] ) and Berger et al., eds. (Guide to ~olecular Clonlng Techni~ues, ~ m1 C Press, Inc., San Diego, CA
[1987] ) .

WO95/11299 2~ ~3S~3 PCTIUS94111716 D. Conqtruct~<-n of Vectors The vectors most useful in practicing this invention are those that are compatible with prokaryotic cell hosts. However, eukaryotic cell hosts, and vectors elt~hle with these cells, are within the scope of the invention .
In certain cases, some of the various vector elements may be already present in commercially lO available vectors such as pUC18, pUCl9, pBR322, the pGEM
vectors (Promega Corp, Madison, WI), the bluescript vectors such as pBIISK+/- (Stratagene Corp., ~a Jolla, CA), and the like, all of which are suitable for prokaryotic cell hosts.
~Iowever, where one or more of the elements are not already present in the vector to be used, they may be individually obtained and ligated into the vector.
Methods used for obtaining each of the elements are well known to the skilled artisan and are comparable to the methods set forth above for obtaining a transgene (i.e., synthesis of the DNA, library screening, and the like).
Preferred vectors of this invention are the pGEM and the bluescript vectors. The most preferred vector is pBIISK+.
Vectors used for amplification of the transgene and/or for transfection of the 1~ n embryos are constructed using methods well known in the art. Such methods include, for example, the standard techni~ues of restriction ~ n~nl1clease digestion, ligation, agarose and acrylamide. gel purification of DNA
and/or RNA, column chromatography purification of DNA
and/or RNA, phenol/chloroform ~ r~lct~n of DNA, DNA
sequencing, polymerase chain reaction amplification, and the like, as set forth in Sambrook et al., supra.
The f inal vector used to practice this invention is typically constructed from a starting 2~ 83~53 WO95/11299 PCr/US94111716 vector such as a commercially avallable vector. This vector may or may not contain some of the elements to be included in the completed vector. If none of the desired elements are present :in the starting vecto~, 5 each element may be individually ligated into the vector by cutting the vector with the appropriate restriction F-ntnnllnl~qe~s) such that~the ends of the element to be ligated in and the ends of the vector are compatible for ligation. In some cases, it may be nen.oC~::/ry to "blunt"
l0 the ends to be ligated together in order to obtain a S3tt cf~,-tory ligation. Blunting is accomplished by first filling in "sticky ends" using Klenow DNA
polymerase or T4 DNA polymerase in the presence of all four nucleotides. This procedure is well known in the 15 art and is described for example in Sambrook et al, supra .
Alternatively, two or more of the elements to be inserted into the vector may f irst be ligated together (if they are to be positioned ad~acent to each 20 other) and then ligated into the vector.
One other method for constructing the vector to conduct all ligations of the various elements simultaneously in one reaction mixture. Here, many nonsense or nonfllnnttnn~l vectors will be generated due 25 to improper ligation or insertion of the elements, however the fllnrttnn~l vector may be identified and selected by restriction ~nt1~1n~l-leAq digestion.
After the vector has been constructed, it may be transfected into a prokaryotic host cell for 30 amplification. Cells typically used for amplification are E col 1 DH5-alpha (Gibco/BRl., Grand Island, NY) and other E. col ~ strains with characteristics similar to DH5-alpha .
Where 1 t ~n host cells are used, cell 35 lines such as Chine5e ham5ter ovary (CHO cells; Urlab et al., Proc. Natl. Acad. Sci USA, 77:4216 [1980])) and 2~83~'j3 Wo 9S111299 PCT/US94/11716 human embryonic kidney cell line 2 93 (Graham et al ., ,J.
Gen. Virol., 36:59 [1977]), as well as other lines, are suitable .
Transfection of the vector into the selected 5 host cell line accomplished uslng such methods as calcium phosphate, electroporation, microinjection, lipofection or DEAE-dextran method. The method selected will in part be a function of the type of host cell to be transfected. These methods and other suitable lO methods are well known to the skilled artisan, and are set forth in Sambrook et al., supra.
After culturing the cells long enough for the vector to be sufficlently amplified (usually overnight for E coli cells), the vector (often termed plasmid at 15 this stage) is isolated from the cells and purified.
Typically, the cells are lysed and the plasmid is extracted from other cell contents. Methods suitable for plasmid purification include ~nter alia, the ~lk~l;n~ lysis mini-prep method (Sambrook et al., 20 supra~ .
er~ration of Plasm;d For Tnqertion into the ~ryo Typically, the plasmid cnrtA~n;n~ the 25 transgene is linearized using a selected restriction ~.n~lnnllnl~qe prior to insertion into the embryo. In some cases, it may be preferable to first isolate the transgene, promoter, and regulatory elements as a linear fragment from the other portions of the vector, thereby 30 in~ecting only a linear nucleic acid sequence enn~A;n;ng the transgene, promoter, intron (if one is to be used), ,~nh;lno~r, polyA se~uence, and optionally a signal se~uence or membrane anchoring domain into the embryo.
This may be accomplished by cutting the plasmid so as to 35 remove the nucleic acid sequence region containing these 2183!~3 elements, and purifying t~his region using agarose gel electroiAhAr~q;~ or other suitable purification methods.
2. Pro~l~Ati~A,n of Tr~n~j~n;c ~ ls The specific line (s) of any mammalian species used to practice this invention are selected for general good health, good embryo yields, good pronuclear visibility in the embryos, and good reproductive 10 fitness. For example, when transgenic mice are to be produced, lines such as C57/B~6 x DBA2 Fl cross, or FVB
lines are often used (obtained commercially from Charles River I.abs, Boston, MA) . The line (s) used to przctice this invention may themselves be transgenlcs, and/or may 15 be knockouts (i.e., mammals which have one or more genes partially or completely suppressed).
The age of the mammals that are used to obtain embryos and to serve as surrogate hosts is a function of the species used, but is readily determined by one of 20 ordinary skill in the art. For example, when mice are used, pre-puberal females are preferred, as they yield more embryos and respond better to hormone in~ections.
Similarly, the male mammal to be used as a stud will normally be selected by age of sexual 25 maturity, among other criteria.
Administration of h, A~ or other chemical compounds may be necessary to prepare the female for egg production, mating, and/or reimplantation of embryos.
The type of hl ?~ fA~Ators and the yuantity used, as 30 well as the timing of administration of the h~A, ?':
will vary for each species of mammal. Such considerations will be readily apparent to one of ordinary skill in the art Typically, a primed female (~.e., one that is 35 producing eggs that can be fertilized) is mated with a stud male, and the resulting fertilized embryos are then WO95111299 218~53 PCrlUS94111716 removed for introduction of the transgene (8) .
Alternatively, eggs and sperm may be obtained from suitable females and males and used for ln vitro fertilization to produce an embryo suitable for 5 introduction of the transgene.
Normally, fertilized embryos are incubated in suitable media until t_e pronuclei appear. At about this time, exogenous nucleic acid comprising the transgene of interest is introduced into the female or l0 male pronucleus. In some species such as mice, the male pronucleus is preferred.
Introduction of nucleic acid may be accomplished by any means known in the art such as, for example, microin~ection, electroporation, or 15 lipofection. Following introduction of the transgene nucleic acid sequence into the embryo, the embryo may be incubated ln vitro for varying amounts of time, or reimplanted into the surrogate host, or both. In v~tro incubation to maturity is within the scope of this 20 invention. One common method is to incubate the emoryos ~n vitro for about 1-7 days, depending on the species, and then reimplant them into the surrogate host.
Reimplantation is accomplished using standard methods. Usually, the surrogate host is anestheti~ed, 25 and the embryos are inserted into the oviduct. The number of embryos lmplanted into a particular host will vary by specles, but will usually be comparable to the number of offspring the species nAtllrAl ly produces.
Transgenic offspring of the surrogate host may 30 be screened for the presence and/or expression of the transgene by any suitable method. Screening is often accomplished by So~1th~rn blot or Northern blot analysis, using a probe that is complementary to at least a portion of the transgene. Western blot analysis using 35 an antibody against the protein encoded by the transgene may be employed as an alternative or additional metl od 21~

for screening for the presence of the transgene product.
Typically, DNA ls prepared from tall tlssue ~about 1 cm is removed from the tip of the tail) and analyzed by Southern analysis or PCR for-~he transgene.
5 Alternatively, the tissues or cells believed to express the transgene at the highest levels are tested for the presence and expresslon of the transgene uslng Southern analysls or PCR, although any tissues or cell types may be used for thls analysls.
Alternatlve or additlonal methods for evaluatlng the presence of the transgene include, without llmltatlon, suitable biochemical assays such as enzyme and/or lmmunological assays, histologlcal stalns for partlcular markers or enzyme actlvlties, and the llke. Analysls of the blood may also be useful to detect the presence of the transgene product ln the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents.
Progeny of the transgenLc mammals may be obtained by mating the transgenic mammal with a sultable partner, or by in vitro fertilizatlon of eggs and/or sperm obtalned from the transgenic mammal. Where mating wlth a partner is to be performed, the partner may or may not be transgenlc and/or a knockout; where lt ls transgenic, it may contaln the same or a dlfferent transgene, or both. Alternatlvely, the partner may be a parental llne. Where in v~tro fertilization is used, the fertilized embryo may be implanted into a surrogate host or incubated ln vltro, or both. Uslng elther method, the progeny may be evaluated for the presence of the transgene uslng methods descrlbed above, or other appropriate methods.
The transgenic mammals of this inventlon may be used to generate one or more cell llnes. Such cell llnes have many uses, as for example, to evaluate the WO95/11299 ~1 8~S5~ PCr/US94111716 effect (s) of the transgene on a particular tissue or organ, and to screen compounds that may affect the level of activity of the transgene in the tissue. Such compounds may be useful as therapeutics to modulate the 5 activity of the transgene.
Production of cell lines may be accomplished using a variety of methods, known to the skilled artisan. The actual culturing conditions will depend on the tissue and type of cells to be cultured. Various lO media containing different concentrations of macro and micro nutrients, growth factors, serum, and the like, can be tested on the cells without undue experimentation to determine the optimal conditions for growth and proliferation of the cells. Similarly, other culturing 15 conditions such as cell density, media temperature, and carbon dioxide concentrations in the incubator can also readily be evaluated.
The transformed mammals, their progeny, and transgenic cell lines of the present invention provide 20 3everal important uses that will be readily apparent to one of ordinary skill in the art. The mammals and cell lines are particularly useful for ~a) providing treatments (such as gene therapy) for a variety of condltlons and dlseases, and/or (b) screening compounds 25 that have potentlal as prophylactics or therapeutics.
Such uses may be found for (l) conditions caused by in~l: t~on, (2) immune system disorders, ~3) epithelial cell repair (skin, lung and/or intestinal epithelia), (4) hematopoiesis, and/or (5) dlsorders 30 caused by varlous physlcal and/or mental stresses.
In the case of transgenic mammals, screening of candidate compounds is conducted by administering the compound(s) to be tested to the mammal, over a range of doses, and evaluating the mammal ' s physiological 35 response to the compound(s) over time. Adminlstration may be by any appropriate means such as, for examp~e, 2183~3 WO 95/11299 PCrNS94111716 ,, ;~.
oral adminlstration, or administration by in~ection, implantation, or transdermal delivery, ~ ron~i1n~ on the chemical nature of the compound being evaluated. In some cases, it may be appropriate to administer the 5 compound in con~unction with other compounds or co-factors that might enhance the efficacy of the compound.
In screening cell lines for compounds useful in treating the above mentioned problems, the compound is added to the cell culture medium at the appropriate 10 time, and the cellular response to the compound is evaluated over time using the appropriate biochemical and/or histological assays. In some cases, it may be appropriate to apply the compound of interest to the culture medlum in con~unction with other compounds or 15 co-factors that might enhance the ef~icacy of the compound .
The invention will be more fully understood by reference to the following examples. They should not be construed in any way as limiting the scope of the 20 present invention.
EX~MPLES
E le 1: Prep~ration of a F~TiP ~romoter/~T-8 Mf-use A_ Pre~ration of D~ C~n~tructs The overall cloning strategy used to prepare the FABP promoter-IL-8 transgene construct is depicted 30 in Figure 2.
The DNA containing the rat intestinal fatty acid binding protein (rFABP) promoter was prepared by PCR amplification. The template used for PCR was Sprague-Dawley rat genomic DNA prepared from tail 35 tissue. An approximately 1.2 kb fragment between nucleotide positions 1 and 1210 of the rFABP sequence ~183~3 Wo 95/11299 PCT/US94/11716 was generated. This sequence, is set forth and numbered according to the Genbank sequence ~Ar~ s~nn Number M18080; Sweetser et al., J. Biol. Chem., 262:16060-16071 [1987] ) . This sequence of 1210 nucleotides is shown in 5 Figure 1. The primers used for amplification of this sequence were complementary to the 5 ' and 3 ' ends of the sequence, and were ~if'S~n~ to create an EcoRI
restriction site at either end of the rFABP promoter fragment. The amplified fragment was inserted into the 10 EcoRI site of p~C19 (New England Biolabs, Beverly, MA) to generate the plasmid FABPTB.
Next, intron 1 of the apolipoprotein E gene was isolated from the vector pHE54 (Simonet et al., J.
Biol. Chem., 268:8221-8229 [1993]) using PCR
15 amplification. The amplified PCR product contained, in addition to the full length sequence of intron 1, a portion of the 3 ' sequence of exon 1 and a portion of the 5 ' sequence of exon 2 of the same gene. The primers used for amplification created a KpnI site at either end 20 of the amplified sequence. The primer sequences were:
Primer 1: CGGAATTccGGAGGTGAAGGAcGTccTTcc (SE~ ID NO: 2) Primer 2: rr~r~AlTTorrATTTGTAGGccTTcAAcTcc (SEQ ID NO: 3 Thirty cycles of ampl ~ f ~ CAt 1 nn were conducted as follows: Denaturation was at 94C for 30 seconds;
annealing was at 54C for 30 seconds; and ~Y~.~n~nn was at 72C for 30 seconds.
The PCR product of about 800 base pairs was digested with KpnI and inserted into KpnI cut FABPTB.
The resulting vector was designated FABP-Eintron. The promoter-ApoE intron cassette was excised from FABP-35 Eintron as an EcoRI fragment and cloned into EcoRI cutpBIISK+ (Stratagene Corp., La Jolla, CA) to generate the ~1835~3 WO 95/11299 PCr/US9~/11716 plasmid pFE-BS. The promoter-intron cassette was subcloned into this vector in both or;~nt~t;~n~ for future use. .
The human IL-8 cDNA was obtained by screening 5 a human peripheral blood lymphocyte cDNA library, prepared as follows:
Peripheral blood lymphocytes were isolated from freshly prepared buffy coats, on a ficol-paque step gradient (Pharmacia, Uppsala, Sweden) . Mf nrln~ e:~r 10 cells present in the interphase of the gradient were removed and washed with PBS three times. The cells were then suspended in the medium RPMI 1640 + 10~ FCS ~fetal calf serum) . About 5 million cells/ml were incubated with poke weed mitogen (10 ug/ml, Sigma Chemical Corp., 15 St. Louis, MO) for 19 hours, followed by addition of cyt~ hP~c;m;~lp to a final concentration of 10 ug/ml for an ~cl~l;t;~nAl ~ hours. Tnrllh~t;~n was carried out at 37C and 5~ CO2.
Total RNA was isolated from activated 20 lymphocytes using the guanidium thiocyanate-CsCl technique ~Chirgwin et al., ~lochem., 18: 5294-5299 [1979]) . Polyadenylated RNA was selected by oligo(dT) chromatography. The polyA+ RNA was then ethanol precipitated and centrifuged. The final pellet was 25 dissolved in water and kept in liquid nitrogen in aliquots .
About 5 ug of polyA+ RNA were used for cDNA
library construction. After denaturation with methyl mercury hydroxide, oligo (dT) -primed double strand cDNA
30 was synthPs; 7P~i following the procedure set forth in Sambrook et al., supra, followed by methylation with Eco RI and Alu methylases. ~he technique of Dorssers et al, (Nuc. Acad. Res., 15: 3629, [1987] ) was used to introduce Eco RI and E~ind III sites on the 5 ' and 3 ' 35 ends of the cDNAs, respectively. After digestion with Eco RI and Elind III restriction enzymes, cDNAs tha~ were WO 95lll299 218 3 S5 3 PCT/US94/11716 larger than 500 base palrs were isolated from an agarose gel by electroelution. The eukaryotlc expression vector Vl9-10 (described above), was digested with Eco RI and Elind III and was then ligated with the cDNAs. These new 5 plasmids containing cDNA inserts were transfected into competent DH5 alpha cells (GIBCO-BR~, Gaithersburg, MD).
The cDNA library was frozen in alir~uots at -80C after addition of DMSO to 7% ~Okayama & Berg, Mol. Cell.
B~ol., 2: 161--170, 1982) .
A mixed oligonucleotlde probe was desLgned on the basis of similarity in nucleotide sequences surrounding and coding for the signal peptidase cleavage site of a number of cytokines. The sequence of this degenerate probe was:
ATGTrr.~ Yr~ T~:yA (SEQ ID NO: 4) In this sequence, M, W, S, V, R, Y, and ~
represent degenerate nucleotides. M represents A or C;
20 W represents A or T; S represents C or G; V represents A
or C or G; R represents A or G; Y represents C or T; and U represents A or C or T.
Using this probe, a cDNA encoding IL-8 was 25 obtained. The I~-8 cDNA clone was seriuenced to confirm that it was homologous with the pl-hl ~ ~h~d se~-~nre (Furutani et al., Biophys. Biochem. Res. Comm., 159:249-255 [1989] ) . This I1-8 cDNA was then used as a template to PCR amplify a SpeI-NotI I'r~; nt of the cDNA.
30 Amplification was accomplished using the following oligonucleotide primers:
Primer 3: GGACTAGTrr~r-~rr~r~rA~rfrTTCTAG (SEQ ID NO: 5) 35 Pr~mer 4: ATAAGAA~ r'r~rTGCATCTGGCAACCC ~SEQ ID NO: 6) 2~835~3 Thirty cycles Or amplification were conducted as follows: Denaturation was at 94C for 30 seconds;
annealing was at 54C for 30 seconds; and extension was at 72C for 30 seconds.
The amplified fragment was then subcloned into SpeI--NotI cut pIIBS--PA (NS) to produce the plasmid pIII--8 PA. The vector pIIBS-PA (NS) was prepared by cloning the SV 40 polyadenylation sequence into the vector pIIBS+ (Stratagene, La Jolla, CA) . The eukaryotic lO expression vector Vl9-lO was used as a template for amplification of the SV40 polyA+ signal. This vector, Vl9-lO, was constructed by inserting a 592 base pair AatII/ClaI fragment containing the origin of repl~rAt~rn seriuence from bacteriophage Ml3 into the eukaryotic expression vector Vl9-8 (described in WO 9l/05795, pllhl~cheri May 2, l99l) . The approximately 242 base pair polyA+ seriuence from Vl9-lO was amplified as a NotI-SacII fragment or a HindIII-XhoI fragment using PCR.
The primers used for PCR amplif ication were:
NotI-SacII frarml~nt:
Primer 5: CTrT~r~ rrrTAATTcAGTc (SEQ ID NO: 7) 25 Primer 6~ A ,~ ~ AArAr,rrr~rArrTCGG ~SEQ ID NO: ô) Thirty cycles of amplification were conducted as follows: DonAtllrAti~ln was at 94C for 30 seconds;
annealing was at 56C for 30 seconds; and P~t.~nc~rn was 30 at 72C for 30 seconds.
~ n~lTII-XhoI fra nt:
Primer 7: CTrlrAr~ r-r~TAATTcAGTc ~SEQ ID NO: 9) Primer 8: CTGGATCTrr~r-r-r~rrrr~r~r7r~'rrAq`~rC ~SEQ ID NO: 10) ~ W0 95/11299 2 ~ ~ 3 7~ ~ rcTNsg4/ll7l6 Thirty cycles of amplification were conducted as follows: Denaturation was at 94C for 30 seconds;
AnneAl 1ng was at 57C for 30 seconds; and extension was at 72C for 30 seconds.
The PCR fragments were sequenced and showed 100% homology to the template. The fragments were then subcloned into NotI-SacII cut or HindIII-XhoI cut pBIISK+, to generate the plasmids pBS--PA (NS) and pBS--PA
~HX), respectively. The amplified IL-8 sequence, which lacked a portion of the 3' llntrAn~l~ted sequence of the original IL-8 cDNA, was sequence verified and found to be 100% homologous to human IL-8 in the codlng region.
To make the final construct for microin~ection of mouse embryos, the promoter-intron casette was excised from pFE-BS as a XhoI-SpeI fragment and subcloned into XhoI-SpeI cut pIL-8PA to generate the plasmid pFE-IL-8 PA, also called FE8.
For microinjection into mouse embryos, the vector was digested with XhoI, ScaI and AflIII, to obtain an approximately 3.1 kb XhoI-AflIII insert fragment cont~1n1ng the rFABP promote~r, a portion of the ApoE first exon, the ApoE first intron, a portion of the second exon, the human IL-8 cDNA and the SV40 polyadenylation signal. This fragment was purified on a 0 . 8% ultrapure DNA agarose gel (BRL Corp ., Bethesda, MD) and diluted to 1 ng/ul in 5mM Tris, pH 7 . 4, 0 . 5mM EDTA.
About 2-3 picoliters of this solution were used for microin jection .
Pregnant mare's serum ("PMS"), supplying Follicle Stimulating Hormone ( "FSH" ) was adminlstered to female mice of the strain BDF1 (Charles River Labs, Boston, MA) about three days prior to the day of microin~ection. PMS (obtained from Sigma Chemicals) was prepared as a 50 I .U. /ml solution in Phosphate Buffered Saline and in~ected intraperitoneally at 0 .1 ml (5 I . U . ) per animal. Human Chorionic Gonadotropin ~"HCG"), 21~3553 supplying Luteinizing Hormone ( "LH" ) was adminlstered 45-48 hours after the PMS in~ections. HCG was also prepared as a 50 I.U./ml solution in PBS and in~ected IP
~intraperitoneally) at 0 . l ml per animal . Females were 5 placed with stud males of the same strain immediately after HCG in~ections. After mating, the females were examined for a vaginal copulation plug. The appearance of an opaque white plug indicated a successful mating.
Successfully mated females were sacrificed by lO cervical dislocation, and both oviducts were rapidly removed and placed in M2 medium (Hogan et al., eds., Manipulating the Mouse Embryo: A Laooratory Manual, Cold Spring Harbor Laboratory Press, pp 249-257 [1986] ) .
The oviducts were transferred individually from M2 15 medium to PBS containing 300 llg/ml hyaluronidase (Sigma Corp., St . ~ouis, MO. ) in a round bottom dissection slide. The embryos were teased out of the oviduct and allowed to settle at the bottom of the slide as the cumulus cells detached from the embryos. When the 20 cumulus masses were disaggregated (about 5 minutes) the embryos were transferred through two washes of M2 medium and the fertilized embryos were separated from unfertilized and abnormal embryos. The fertilized embryos were then transferred through 5~ CO2 25 equilibrated Ml6 medium (Hogan et al., supra), placed in eq~ hrA~ed microdrop dishes containing Ml6 medium under paraffin oil and returned to the ~nc-lhat~r.
Fertilized single-cell embryos from BDFl xBDFl-bred mice were selected in Ml6 medium and 30 incubated about 5 hours at 37C until the pronuclei appeared. Embryos were then transferred into M2 medium in a shallow depression slide under paraffin oil and placed under the microscope. The pronuclei were easily visible under 200X magnification. Using suction on the 35 holding pipet, a single embryo was selected and rotated such that the male pronucleus was away from the holding _ 3~ PCI/US94/11716 pipet. Approximately 2 to 3 picoliters of solution rnnt;~;n;ng the DNA construct at about l microgram per ml was in~ected lnto one of the pronucleil preferably the male pr~n~lc~ q~ Following the injection, the embryos 5 were returned to incubation for 18 hours and reimplanted the next day into foster pseudopregnant females.
Reimplantations were performed on anesthetized female mice of strain CDl using a dissecting microscope.
A pseudo-pregnant female mouse was anaesthetized with 0.017-0.020 ml/g body weight of avertin, injected IP.
The mouse was placed under the dissecting mlcroscope and the incision area was disinfected with 70% ethanol. The ovary was exteriorized and the bursal sac that surrounds the ovary and the oviduct was carefully pulled open.
15 The ovary and oviduct were separated to expose the opening of the oviduct (termed the inf indibulum) .
Surviving embryos were then removed f rom the incubator and loaded into the reimplantation pipet. The tip of the pipet was inserted several m~ 11; ters into the 20 ;nf;n~;hulum and gentle pressure was used to deliver the embryos into the oviduct. About lO to 20 2-cell embryos were implanted per mouse, resulting in a litter size of about 3 to 12. The ovary then was returned to the peritoneum, and the body wall and then the skin were 25 sutured.
B. Ifl~ontification of Tr~n~n1c ~I;ce Of 56 mice born after embryo injections, ll 30 ~ ~nf :1; n~d the IL-8 transgene as assayed by PCR
amplification. About l cm of the tail of each mouse was removed, and DNA was prepared using the technique set forth by Hogan et al., supra. The DNA was then sub~ected to PCR analysis using the following primers:

2183553 .
W095111~99 PCTIUS94111716 Primer 9: GCCTrTAr~AAAri~rr~rr7rirAr (SEQ ID NO: 11) Primer 10: ~ A'r'r'rATGAt~C iSE9 ID NO: 12~
The PCR amplification procedure was denaturation at 94C for 30 seconds, ~nn~Al ~nS at 56C
for 30 seconds, and ~t~nq1rn at 72C for 30 seconds.
Thirty cycles were performed.
The resultant transgenlc mice harboring the transgene in their genome are termed the founder mice.
The founder mice were b~rk~r~ssed to strain BDF1 mice to generate heterozygous F1 transgenic mice.
To evaluate the F1 transgenic mice for the presence and effect of IL-8, blood was obtained and analyzed as follows.
Quantitation of serum IL-8 levels were determined using an Elisa kit for human IL-8 (obtained from Biosource International, Camarillo, CA) and following the manufacturer's protocol. The results are shown in Figure 3 The serum level of IL-8 in two lines of transgenics is shown in Figure 3A. These mice have levels of IL-8 of between about 5 and 15 ng/ml blood, whereas no IL-8 is detectable in the non-transgenic (NT) control mice.
Circulating white blood cells in the serum of the F1 transgenic and non-transgenic mice were counted using a Sysmex F-800 blood cell counter (Toa Medical Electronics Co., LTD, Kobe, Japan) and following the manufzcturer ' s protocol . Prior to counting, red blood cells were lysed with QuicklyserTM (Toa Medical Electronics Co., LTD, Kobe, Japan), following the manufacturer's protocol. For differential leukocyte analysis, about 3 ~Ll of whole blood were spread on a glass slide and subjected to Wright's-Giemsa staining.
At least 100 cells were counted from each slide by v~ 7~ng the cells under a lOOx oil emersion lens on WO 9S/11299 ~ L 8 3 ~5 3 PCT/US94~11716 .

an Olympus CH2 student microscope. Neutrophils were distinguished from lymphocytes, macrophages, Prc;nnrh;ls, and bAcophllc by their mul~;ntlr~l~Ated structures. For all lines reported, at least five S individual Fl heterozygotes were bled and analyzed.
Absolute neutrophil levels were determined by multiplying the percentage of neutrophils on the Wright ' s-Giemsa stained slides by the total white blood cell count obtained from the Sysmex counter. The results are shown in Figure 3B. The level of neutrophils in the transgenic mice is substantially higher than that of the non-transgenic (NT) control mice .
E le 2: Prel;1aration of a F~RP ~r ter/R(~ Mrllce The overall cloning strategy used to prepare the FABP promoter KGF transgene construct is depicted in Figure 2.
An expression vector for use with a variety of transgenes was generated by digesting the plasmid pFE-BS
~described in Example l) with the restriction rn~lrn~lrl eases XhoI and SpeI and isolating the fragment containing the rat FABP promoter-ApoE intron ser~uence ~3 ' portion of exon l, full length sequence of intron l, and 5 ' portion of exon 2; described in Example l) . This ca3sette was then inserted into the vector pIIBS-PA
~NS), which is described in Example l. The resulting vector, which contains the rat FABP promoter and ApoE
se~uence upstream of a polylinker and SV40 polyadenylation site was designated pFEPA~3.
The gene encoding human RGF ~keratinocyte growth factor) was obtained by PCR amplification of the gene from a normal human dermal fibroblast cDNA library.
PCR amplification of KGF was accomplished using the following two oligonucleotide primers:

~1835~3 WO 95111299 PCT/US94~11716 Primer 11: CAATCTACAATTCACAGA (SEQ ID NO: 13) Primer 12: TTAAGTTATTGCCAT~GG (SEQ ID NO: 14) ,~ .~';, .' 5 The conditions for PCR were. denaturation at 92C for 20 seconds; anneal at 55-40C for 20 seconds ~this consisted of 2 cycles at 55C, followed by 2 cycles at 45C, which was followed by 28 cycles at 40C); and extension at 72C for 30 seconds. Thirty cycles total 10 were performed.
To lntroduce HindIII and BglII restriction sites to the ends of the KGF cDNA, the cDNA was PCR amplified using the following two oligonucleotide primers:
15 Primer 13: A;~CAAAGCTTCTACAAT'rrA~'Ar.ArA~ A (SEQ ID NO: 1~) Primer 14: AACAAGATCTTAAGTTATTGCCATAGG (SEQ ID NO: 16) The conditions for PCR were: denaturation at 92C for 20 20 seconds; anneal at 45C for 20 seconds; and elongation at 72C for 30 seconds. Thirty cycles were performed.
After amplification, the KGF cDNA was purified and digested with HindIII and BglII, and then ligated into the vector pCFM3006. This vector was prepared from 25 the vector pCFM836 (described in U.S. Patent No.
4,710,473, issued December 1, 1987). The two endogenous NdeI restriction sites in pCFM836 were removed by cutting pCFM836 with NdeI, filling in the cut ends of the vector using T4 polymerase, and then re-l i~t~n~ the 30 vector by blunt end ligation. Next, the DNA sequence between the AatII and KpnI sites of the now modified pCFM836 was altered using the technique of PCR
overlapping oligonucleotide mutagenesis. The following changes at the base pair positions listed were made ~the 35 base pair position changes are relative to the BglII
site on pFM836 which is position #180):
_ . . _ .. .. . . . _ . .. . . . . .. .. .. .. ..

WO 95111299 ~ ~ 8 3 ~5 3 PCT/US94~1171C
.
-- 3!~ --m; d h~ $ b~ ch A n Sr~d # 428 G/C
# 509 A/T
# 617 insert two G/C bp $ 978 C/G
# 992 A/T
# 1002 C/G

# 1026 T/A
# 1045 T/A
# 1176 T/A
# 1464 T/A
# 2026 bp deletion # 2186 T/A
# 2479 T/A
# 2498-2501 ~
# 2641-2647 bp deletion # 3441 A/T
# 3 64 9 T/A

The KGF cDNA in t~is vector was used as a template for amplification. A 710 base pair HindIII
fragment of KGF was amplified using PCR and the 25 following two oligonucleotide primers:
Pr~er 15: CGATCGTAAGCTTGGTCAAT(~'rT~rr-At~ r ~SEQ ID NO: 17) Primer 16: CGATCGTA~GCTTG-Crfi1~CrT~r~TTATTGCC (SE:Q ID NO: 18) Ampliflcation was conducted for 30 cycles. Denaturation was at 94C for 30 seconds, ~nn.o~l;ng was at 58C for 20 seconds, and elongation was at 72C for 30 seconds. The amplified fragment was puri~ied by agarose gel 35 electrophoresis, and then was ligated into the vector pFEPA#3 which had been previously digested with XbaI.

WO 95111299 ~ 1 8 3 5 5 3 PCTIUS94/11716 The ligated vector containing the KGF insert was transformed into E coli DE15 alpha cells, and colonies of the cells grown overnight on agarose plates were then evaluated by restriction digest of the plasmids to identify those that harbored plasmid with KGF in the proper orientation Cells containing the proper plasmid were isolated and amplified by culturing overnight. After culturing, the plasmid was purified using the ~lk~l~ne lysis method along with CsCl gradient centrifugation. The purified plasmid was designated FEK .
For microinjection into mouse embryos, the plasmid FEK was digested with XhoI, ScaI, and AflIII to obtain a 3.3 kb XhoI-AflIII fr~; ~n~ r~nt~n;n~ the rFABP promoter, a portion of the ApoE f irst exon, the ApoE first intron, a portion of the second exon, the human KGF cDNA and the SV40 polyadenylation signal.
This fr~; ~ ' was purified on a 0.8% ultrapure DNA
agarose gel (BRL Corp., Bethesda, ND) and diluted to l ng/ul in 5mM Tris, pH 7.4, 0.5mM EDTA. About 2-3 picoliters of this solution were used for microinjection. Microinjection and implantation into pseudopregnant mice were conducted as described in Example l.
Of 77 mice born, 14 contained the transgene as analyzed by PCR, using the same methods and probes as for analysis of the IL-8 transgenic mice in Example l.
All literature cited herein is spe~ ~f~c~lly incorporated by reference.

2~83~S3 SEQUENCE LISTING
~1 ) GENERAL INFORMATION:
~i) APPLICANT: Amgen Inc.
~ii) TITLE OF INVENTION: Enhanced Transgene Expression In Specific TissueS
~iii) NUM~BER OF SEQUENCES: 18 ~iv) Cu~;~l~l ADDRESS:
IA) ~nn~Cc~ Amgen Inc.
(B) STREET: Amgen Center 1840 Dehavilland Drive (C) CITY: Thousand Oaks (D) STATE: California (E) COUNTRY: USA
(F) ZIP: 91320-1789 (v) COMPUTER READABLE FORM
(A) MEDIU~ TYPE: Diskette, 3.5 in., DS, 2.0 ~b (B) COM~PUTER: Apple MAr~n~lsh (C) OPERATING SYSTEM: MA~ ~ n~ h oS 7 0 (D) SOFTWARE: Microso~t Word Version 5.1a (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 18--OCT-1593 (C) CLASSIFICATION:
(2) INFORMATION FûR SEQ ID NO:1:
(i) SEQUENCE rRA~ RT~TICS:
(A) LENGTH: 1210 base pairs (B) TYPE: nucleic acid (C) ST~ C~i double stranded (D) TOPOLOGY: linear (xi ) SEQUENCE U~:~U~~ N: SEQ ID NO :1:

~g3~3 GAATTCCTTA ATTTGQTA/L TTTACTQQ TTAGTCAAGA AQ~AAQTT 50 T~A~AA~AT~ A~r.ArçTArr TGATATGTAA ATACTGAAAA QQTTCGGT l00 GAAAAGATTC AAArATAA~A Ar~rTArrTG ATATATAATT ATATTTGTAT 150 GAAATGTCQ TAr.~r.r.TAr.A A~r~TGr~ATRr~ GAACCTA~TG GTCTAAAAAT 200 AGAAAATGAG AGCTGGAGAG ATGGCTQGC AGTTAAAAGC ACTGACTGCT . 250 CQTCTGTAA TGGGATCTGA ll.~ ,L~.LLI., l~l~lL~LI~ A~r~rAr.rr, 350 GTGGTGTACT rAr~TArP~TA AAr.TAZ~APlAA TTCTTTAAAA ~A~ TAr.~ 400 ACTAAATTTT AGATTGATTT GATATQATA AAATAACCTT ~rr~ r~rT~r~ 550 TTACATAATT AQTAGTTAC ACCTGCTGTA r.Ar.Tr~T~:r~r TTTGAACCTG 750 T~A~TArAAA AGTATCTTGA ~ lLLLLL~, LL~,lLVLL~ lLLLVL~_L~ 800 L LL~JLLL~ TTTATCGCCT GATTQTTCT l~:lLL~:lLLL`JL ~ - LLLL~ L~J 850 GAGTGGAACT CCTTATTAQ rrArGrTAr~ ATTGTCTGCC L'_L~ ,LL~_L~ 900 GAGAGCTCGG ATTAAAGGTG r.~Ar.crATrA QCTTGACCC TAATTCTTGG 950 Al~TAl~Al~TG CCTAQTGCT GTAGTCGGAG ~r~rAr.T~r.r. TATGGTTACC l000 AAATTTGAAT GQGTTGAAT rTrArtrAAT~ GATTQAAGA ~Ar.rArTArA 1050 ArllrAA~rTA A~rr.r.r.rCTr. çr~Tr'r~ CTGCQGGTT ATCTCTTGAA ll00 CTTTGAACTT CQQTQTG GTATGAATTG GTTCGAAGAT A~rAl~T~r.l~ 1150 ATAAATTCTC TCTAGTGGAC A~r~Arrr~AT ~L~L~i~LLL~. rTAr.~rrr~r 1200 (2) INFOR~5ATION FOR SE:Q ID NO:2:

2183~

(i~ SEQUENCE t~RARA(~TrRrqTICS
~A) LENGTH: 30 ba3e PairS
(B) TYPE: nUC1eiC aCid (C) STRPI~TnP~r~CS aing1e Stranded (D) TOPOLOGY: 1inear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
CGGAATTCCG r~"'`.Tr.AAt:~ ACGTCCTTCC 30 (2) INFORI~ATION FOR SEQ ID NO:3:
(i) SEQUENCE r~I~O~T~RTSTICS
(A) LENGTH: 30 ba3e PairS
(B) TYPE: nUC1eiC aCid (C) STRANn~nNl:~cs ~ingle Ctranded (D) TOPOLOGY: 1inear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

(2) INFO.NATION FOR SEQ ID NO:4:
(i) SEQUENCE (`RARArTF:RT.CTTf'C
(A) LENGTH: 30 ba~e Pair:l (B) TYPE: nUC1eiC aCid (C) STP~ ing1e 5tranded (D) TOPOLOGY: 1inear (Yi ) SEQUENCE ~ 5~ 10N: SEQ ID NO: 4:
ATGTCGAr~W C:~VL5~ C~ YA 30 (2) INFORI`IATION FOR SEQ ID NO:S:
(i) SEQUENCE ~ ARA~T~RrqTICS
(A) LENGTH: 29 b~-~e Pair9 (B) TYPE: nUC1eiC aCid (C) STRA~ n-.r~.C Sing1e Stranded (D) TOPOLOGY: 1inear 2183~3 WO9~/11299~ PCrlUS94/11716 (xi) SEQUENC~ DESCRIPTION: SE9 ID NO:5:
GGACTAGTCC Ar.~rr~r~r~ AGCTTCTAG ~ ~ 29 (2) INFORXATION FOR SEQ ID NO:6:
(l) SEQUENCE rR~R~rT~RTSTTCS:
(A) LENGT~: 39 base pair~
(B) TYPE: nucleic acid (C) STP~ `S: single 3tranded (D) TOPOLOGY: linear (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
r~TGr rr~rrGrT~ CTATTGQTC TGGQACCC 39 (2) INFORNATION FOR SEQ ID No:7:
(i) SEQUENCE rR~R~rTr~RTsTTrq (A) LENGTH: 22 base pAir~
(B) TYPE: nuclelc acid (C) STR~ ~c single stranded (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CTCTAGAAAG CTTA~TTCAG TC 22 (2) INFORNATION FOR SEQ ID NO:8:
(i) SEQUENCE rR~R~rT~RTcTTrq (A~ LENGT~R: 2 8 ba3e pair3 (B) TYPE: nucleic acid (C) STP~ : sinyle stranded (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
r~r.~r.rGr~ GAGCTCGG 28 (2) INFORNATION FOR SEQ ID NO:9:

2t~3~3 (i) SEQUENCE ~ AR~'T~:RTSTICS:
(A) LENGTH: 22 ba3e pair:~
(B) TYPE: nucleic acid (C) STR~ ingle 3tranded (D) TOPOLOGY: linear (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

(2) INFORMATION FOR SEQ ID NO:l0:
(i) SEQUENCE rRARArT~RT~TTOE
(A) LENGTH: 31 ba3e paira (B) TYPB: nucleic acid (C) STRANn~n'`T~qC: aingle 3tranded (D) TOPOLOGY: linear (xi) SEQUENOE DESCRIPTION: SEQ ID NO:l0:
CTGGATCTCG A(~TAI-rf q~. GGATCATAAT C 31 ~2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE ~TTARAt~T~RrcTIcs:
(A) LENGTH: 21 ba3e pair~
(B) TYPE: nucleic acid (C) STR~ : 3ingle 3tranded (D) TOPOLOGY: line~r (xi) SEQUENCE l~I:.5~ JN: SEQ ID NO:ll:

(2) INFORNATION FOR SEQ ID NO:12:
(i) SEQUENOE ~ ARAt~T~RT~cTIcs:
(A) LENGTH: 21 ba3e pair3 (B) TYPE: nucleic acid (C) STPI~ ~: 3ingle 3tranded (D) TOPOLOGY: linear 218~3 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: , C~ c CATTTATGAG C .', 21 (2) INFORMATION FOR SEQ ID NO:13: .
(i~ SEQUENCE rRAR~'`T~RTSTT~S:
(A) LENGTH: 18 baae pairs (R) TYPE: nucleic acid (C) STRA : single atranded (D) TOPOLOGY: linear Ixi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

~2) INFORMATION FOR SEQ ID NO:14:
~1) SEQUENCE t'RA~'TERTCTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid ~C) SrRANnrnN~: single stranded ~D) TOPOLOGY: linear ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

(2) INFORMATION FOR SEQ ID NO:lS:
(i) SEQUENCE f~RAQA(`TERT~TICs:
(A) LENGTH: 29 base pairY
(B) TYPE: nucleic acid (C) cTRZ~ ~~: single stranded (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:lS:
AAC~AAGCTT CTACAATTCA CAGATAGGA 2 9 ~2) INFORMATION FOR SEQ ID NO:16:

~183~3 WO 95/11299 PCTrUS94rll716 (i) SEQUENCE rRDRDrlr~RTc~rTcs (A) LENGTH: 27 base palr~
(B) TYPE: nucleic ~cid (C) .CTPDr- ~n~ S ~ingle stranded (D) TOPOLOGY: linear (xi) SEQUENOE DESCRIPTION: SEQ ID NO:16:

12 ) INFORMATION FOR SEQ ID NO :17:
(i) SEQUENCE rHDR~rTF:RT.CTIcs (A) LENGTH: 34 base pair~
(B) TYPE: nucleic acld (C) S~RD : ~lngle atranded (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
CGATCGTAAG CTTGGTC~AT r.ArrTDr~Dr. TAAC 34 (2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE rRDRDrT~RTcTIcs (A) LENGTH: 33 base pair~
(B) TYPE: nucleic acid (C) Sr'pD~ cS ~ingle ~tranded (D) TOPOLOGY: linear (xi) SEQUENCE L/~S~K~ ON: SEQ ID NO:l~:

Claims (19)

We Claim:

1. A nucleic acid sequence comprising at least a portion of a transgene, excluding human growth hormone, beta-galactosidase, and SV40 T antigen, wherein the transgene is operably linked to a promoter selected from the group consisting of: intestinal FABP promoter, liver FABP promoter, and ApoC-III promoter.

2. The nucleic acid sequence of claim 1 further comprising a polyadenylation sequence.

3. The nucleic acid sequence of claim 2 further comprising an intron.

4. The nucleic acid sequence of claim 3 wherein the transgene comprises a nucleic acid encoding a polypeptide involved in the immune response, hematopoiesis, inflammation, cell growth and proliferation, cell lineage differentiation, or the stress response.

5. The nucleic acid sequence of claim 4 wherein the transgene is selected from the group consisting of: interleukin 1, interleukin 2, interleukin 3, interleukin 4, interleukin 5, interleukin 6, interleukin 7, interleukin 8, interleukin 9, interleukin 10, interleukin 11, interleukin 12, ENA-78, interferon-.alpha.
interferon-.beta., interferon-.gamma., granulocyte-colony stimulating factor, granulocyte-macrophage colony stimulating factor, macrophage colony stimulating factor, stem cell factor, keratinocyte growth factor, MCP-1 and TNF, and fragments thereof.

6. The nucleic acid sequence of claim 5 comprising the rat intestinal FABP promoter, the human ApoE intron 1 linked at its 5' end to the 3' portion of the human ApoE exon 1 and at its 3' end to the 5' portion of the human ApoE exon 2, and the coding sequence of the transgene human IL-8.

7. The nucleic acid sequence of claim 4 comprising the rat intestinal FABP promoter, the human ApoE intron 1 linked at its 5' end to the 3' portion of human ApoE exon 1 and at its 3' end to the 5' portion of the human ApoE exon 2, and the coding sequence of the transgene human KGF.

8. A non-human mammal and its progeny containing a nucleic acid sequence comprising at least a portion of a transgene excluding human growth hormone, beta-galactosidase, and SV 40 T antigen, operably linked to a promoter selected from the group consisting of:
liver fatty acid bind protein promoter, intestinal fatty acid binding protein promoter, and ApoC-III.

9. The mammal of claim 8 wherein the promoter is rat fatty acid binding protein promoter.

10. The mammal of claim 9 wherein the nucleic acid sequence further comprises an intron.

11. The mammal of claim 10 wherein the transgene comprises a nucleic acid encoding a polypeptide involved in the immune response, hematopoiesis, inflammation, cell growth and proliferation, cell lineage differentiation, or the stress response.

12. The mammal of claim 11 wherein the nucleic acid sequence comprises the rat intestinal FABP
promoter, the human ApoE intron 1 linked at its 5' end to the 3' portion of the human ApoE exon 1 and at its 3' end to the 5' portion of the human ApoE exon 2, and the transgene human IL-8 or KGF.

13. The non-human mammal of claims 8, 9, 10, 11 or 12 that is a rodent.

14. The rodent of claim 12 that is a mouse.

15. A vector comprising the nucleic acid sequence of claims 1, 2, 3, 4, 5, 6, or 7.

16. A eukaryotic cell containing the nucleic acid sequence of claims 1, 2, 3, 4, 5, 6, or 7.

17. A prokaryotic cell containing the nucleic acid sequence of claims 1, 2, 3, 4, 5, 6, or 7.

18. A eukaryotic cell containing the vector of claim 15.

19. A prokaryotic cell containing the vector of claim 15.