WO1995011299A1 - 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

ENHANCED TRANSGENE EXPRESSION IN SPECIFIC TISSUES OF THF GASTROINTESTINAL TRACT

Field of the Invention

This invention relates to the field of recombinant DNA technology, especially to nucleic acid sequences useful for constructing a transgenic mammal . More specifically, the invention concerns expression of a transgene in certain tissues or organs of a non-human mamm l .

Description of Related Art

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 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 offspring of the surrogate host are evaluated for the presence of the transgene.

Expression of the transgene, i.e., production of the protein encoded by the transgene nucleic acid sequence, may confer a new phenotype on the mammal. 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 in vivo screening and testing of compounds that may be useful in treating or preventing the disease (s), and/or for developing methods useful in diagnosing 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 primarily 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. This promoter, containing about 1.2 kb of 5' flanking sequence, has been demonstrated to confer lineage specific expression of certain transgenes in the gastro-intestinal villus enterocytes of mice. The transgenes evaluated include, for example, human growth hormone and SV40 T-antigen (Rottman et al . , J. Biol . Chem. , 268:11994-12002 [1993]; Hauft et al . , Clin . Res . , 39.325A [1991]; Roth et al . , Proc . Natl . Acad . Sci USA, 88:9407-9411 [1991]; Roth et al . , Amer. J. Physiol . , 63:G186-G197 [1992]; Cohn et al . , J. Cell . Biol . , 119:27-44 [1992]; Kim et al . , Surg . Forum, 43:153-155 [1992]) . 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 of the interleukins, IL-8 (also known as Neutrophil Activating Peptide-1, or NAP-1) , has been shown to be a neutrophil chemoattractant with the ability to activate neutrophils and stimulate the respiratory burst (Colditz et al . , J. Leukocyte Biol . , 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 inflammation.

Zwahlen et al . { Int . Rev. Exp . Path . , 34B:22- 42 [1993]) describe some effects of IL-8 injected into some rodents. When injected intradermally into rats, IL-8 induced neutrophil infiltration at the site of injection. Intravenous injection of IL-8 into rabbits resulted in neutrophil sequestration in the lungs. Vogels et al . (Antimicrobial 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. Sci . , 629:422- 424 [1991]) show that guinea pigs injected with IL-8 had a higher level of T-lymphocyte and eosinophil accumulation 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 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, published August 9, 1990) .

Guo et al . (EMBO J. , 12:973-986 [1993]) have prepared a transgenic 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-1 has been cloned and sequenced (Furutani et al . , Biochem . Biophys . Res . Comm . , 159:249- 255 [1989]; Yoshimura et al . , Chemotactic Cytokines, Westwood et al . , eds . Plenum Press, NY [1991], pp.47- 56) . MCP-1 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 injury (Leonard et al . , Immunol . Today, 11:97-101 [1990]) . MCP-1 has been shown to be produced by some tumor cells in vitro, and in human metastatic melanomas in 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 in vitro systems, there is a need in the art to provide in vivo systems to more accurately evaluate the role of these genes in disease.

Accordingly, it is an object 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. It is a further object 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 of ordinary skill in the art.

SUMMARY 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 FABP promoter, liver FABP promoter, and ApoC-III promoter.

In another aspect, the 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 FABP promoter, liver FABP promoter, and ApoC- III promoter, and wherein the transgene is selected from the group of transgenes 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-α interferon-β, interferon-γ, 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. In still another aspect, the invention provides a nucleic acid sequence 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 IL-8 or human KGF.

In one other aspect, the invention provides a mammal or its progeny containing a nucleic acid sequence comprising at least a portion of 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.

In yet another aspect, the invention provides a mammal wherein the nucleic acid sequence comprises the rat intestinal FABP promoter, the human ApoE intron 1 linked at its 5' end to the 3¹ portion of the human ApoE exon 1 and at its 3' end to the 5¹ portion of the human ApoE exon 2, and the transgene human IL-8 or KGF.

In still one other aspect, the invention provides a eukaryotic cell containing a nucleic acid sequence set forth above.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the rat fatty acid binding protein promoter (FABPp) sequence obtained from the full length FABP sequence as it appears in Genbank (accession number M18080) . Figure 2A-C depicts the overall cloning strategy for preparation of the constructs used to make the IL-8 and the KGF transgenic mice. "FABP-p" refers to the rat fatty acid binding protein promoter sequence; "ApoE*" refers a nucleic acid sequence containing the human ApoE DNA sequence encoding the 3' portion of exon 1, the entire sequence of intron 1, and the 5' portion of exon 2; "SV40PA" refers to the SV40 polyadenylation sequence.

Figure 3 depicts the level of IL-8 and the level of circulating neutrophils in both control and transgenic mice. Figure 3A shows serum IL-8 levels. Figure 3B shows circulating neutrophil levels. NT represents non-transgenic (control) mice. The numbers refer to individual lines of transgenic mice used in the analysis.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term "operably linked" refers to the arrangement of various nucleic acid sequence elements relative 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 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 affect the level of expression of the transgene. By modulate is meant increasing, decreasing, or maintaining the level of activity of a particular element. The position 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 referenced by the number of 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 which the nucleic acid sequence is inserted. The term "transgene" is meant to include (1) 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 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 silent naturally occurring or homologous nucleic acid sequence whose expression is induced in the cell into which it has been inserted. By "mutant form" is meant a nucleic acid sequence that contains one or more nucleotides that are different from the wild-type or naturally occurring sequence, i.e., the mutant nucleic acid sequence contains one or more nucleotide substitutions, deletions, and/or insertions. In some cases, the transgene may also include a sequence encoding a leader peptide or signal sequence such that 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 may function alone to regulate transcription, or, in some cases, may act in concert with one or more other 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 ia, such as, for example, mouse, rat, hamster, squirrel, or beaver.

The term "progeny" refers to all offspring of the transgenic mammal, and includes every generation subsequent to the originally transformed transgenic mammal.

Preparation of the Invention

1. Preparation of DNA Constructs

A. Selection of Transgene

This invention contemplates expression of one or more transgenes primarily in certain of the gastro- intestinal tissue of a transgenic mammal. Included 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 sequence encoding a polypeptide involved in the immune response, inflammation, cell growth and proliferation, cell lineage differentiation, and/or the stress response. The transgene 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 thereof that has at least some biological activity.

Optionally, the transgene may be a hybrid nucleic acid sequence, i.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 naturally occurring cDNA and/or genomic sequences . 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 Sambrook et al . (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [1989]) and in Berger and Kimmel {Methods in Enzymology: Guide to Molecular Cloning Techniques, 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 . Int . Ed. Engl . , 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 transgene 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. Where 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 sequence of the probes or primers 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 functional 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 synthesized.

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 (IL-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-α, interferon-β, . interferon-γ, 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 (KGF) , monocyte chemoattractant protein-1 (MCP-1; Furutani et al . , supra) , tumor necrosis factor (TNF) , and fragments, subunits or mutants thereof. More preferred transgenes include erythropoietin, interleukin 8, MCP-1, keratinocyte growth factor, and ENA-78. The most preferred transgenes include human interleukin 8, human keratinocyte growth factor, and MCP-1.

B. Selection of Regulatory Elements

This invention contemplates the use of promoters that enhance transgenic expression in some gastro-intestinal tissues. These promoters may be homologous or heterologous to the transgene and/or to the transgenic mammal. Thus, the promoters used to practice this invention may be obtained from any source. Preferred promoters of this group include the intestinal fatty acid binding protein promoter (FABP promoter) , the liver FABP promoter, and the ApoC-III promoter. The most preferred promoter of this group is the rat intestinal FABP promoter.

The promoter sequences of this invention may be obtained by any of sever-al 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 endonucleases. In some cases, the promoter may have been sequenced. For those promoters whose DNA sequence is known, the promoter may be synthesized using the methods described above for transgene synthesis.

Where all or only portions of the promoter 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 fragment of DNA containing the promoter may be isolated from a larger piece of DNA that may contain, for example, a coding sequence or even another gene or genes. Isolation may be accomplished by restriction endonuclease digestion using one or more carefully selected enzymes to isolate the proper DNA fragment. After digestion, the desired fragment is isolated by agarose gel purification, Qiagen column or other methods known to the skilled artisan. Selection of suitable enzymes to accomplish this purpose will be readily apparent to one of ordinary skill in the art.

C. Selection of Other Vector Components

In addition to the transgene and the promoter, the vectors useful in this invention typically contain one or more other elements useful for (1) optimal functioning of the vector in the mammal into which the vector is transfected, and (2) amplification of the vector in bacterial or mammalian host cells. Each of these elements will be positioned appropriately in the vector with respect to each other element so as to maximize 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 Sequence Element

For those embodiments of the invention where the transgene is to be secreted, a signal sequence, is 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 identified, and any of them that are functional in the transgenic tissue may be used in conjunction with the transgene. Therefore, the signal sequence 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 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. Membrane Anchoring Domain Element

In some cases, it may be desirable to have a transgene expressed on the surface of a particular intracellular membrane or on the plasma membrane. Naturally occurring membrane 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 to confer this feature. Frequently, 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 may first be placed into the vector in the appropriate position as a separate component from the transgene. As for the signal sequence, the anchor domain may be from any source and thus may be homologous or heterologous with respect to both the transgene and the transgenic mammal. Alternatively, the anchor domain may be chemically synthesized using methods set forth above.

iii. Origin of Replication Element

This component is typically a part of prokaryotic expression vectors purchased commercially, and aids in the amplification of the vector in a host cell. If the vector of choice does not contain an origin of replication site, one may be chemically synthesized based on a known sequence, and ligated into the vector.

iv. Transcription Termination Element

This element is typically located 3 ' to the 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 a vector, it can also be readily synthesized using methods for nucleic acid synthesis such as those described above.

v. Intron Element

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 sequence, especially where the 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 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 the intron is 3' to the transcription start site, and 5' to the polyA transcription termination sequence. Preferably for cDNA transgenes, the intron 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, 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 may be used in the vector. A preferred intron is intron 1 of the human ApoE gene.

vi. Selectable Marker ( B ) Element-

Selectable marker genes encode proteins necessary for the survival and growth of transfected cells grown in a selective culture medium. Typical selection 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 Bacilli .

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 . {Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [1989]) and Berger et al . , eds. {Guide to Molecular Cloning Techniques, Academic Press, Inc., San Diego, CA [1987]) . D. Construction 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 compatible 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 available vectors such as pUC18, pUC19, pBR322, the pGEM vectors (Promega Corp, Madison, WI) , the bluescript vectors such as pBIISK+/- (Stratagene Corp., La Jolla, CA) , and the like, all of which are suitable for prokaryotic cell hosts. However, 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 mammalian embryos are constructed using methods well known in the art. Such methods include, for example, the standard techniques of restriction endonuclease digestion, ligation, agarose and acrylamide gel purification of DNA and/or RNA, column chromatography purification of DNA and/or RNA, phenol/chloroform extraction of DNA, DNA sequencing, polymerase chain reaction amplification, and the like, as set forth in Sambrook et al . , supra . The final vector used to practice this invention is typically constructed from a starting vector such as a commercially available 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 vector, each element may be individually ligated into the vector by cutting the vector with the appropriate restriction endonuclease (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 necessary to "blunt" the ends to be ligated together in order to obtain a satisfactory 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 art and is described for example in Sambrook et ai, supra .

Alternatively, two or more of the elements to be inserted into the vector may first be ligated together (if they are to be positioned adjacent to each 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 nonfunctional vectors will be generated due to improper ligation or insertion of the elements, however the functional vector may be identified and selected by restriction endonuclease digestion.

After the vector has been constructed, it may be transfected into a prokaryotic host cell for amplification. Cells typically used for amplification are E coli DH5-alpha (Gibco/BRL, Grand Island, NY) and other E. coli strains with characteristics similar to DH5-alpha.

Where mammalian host cells are used, cell lines such as Chinese hamster ovary (CHO cells; Urlab et al . , Proc . Natl . Acad. Sci USA, 77:4216 [1980])) and human embryonic kidney cell line 293 (Graham et al . , J. Gen . Virol . , 36:59 [1977]), as well as other lines, are suitable.

Transfection of the vector into the selected host cell line accomplished using 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 methods are well known to the skilled artisan, and are set forth in Sambrook et ai. , supra .

After culturing the cells long enough for the vector to be sufficiently amplified (usually overnight for E coli cells), the vector (often termed plasmid at 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 inter alia, the alkaline lysis mini-prep method (Sambrook et al. , supra) .

E. Preparation of Plasmid For Insertion into the Embryo

Typically, the plasmid containing the transgene is linearized using a selected restriction endonuclease 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 injecting only a linear nucleic acid sequence containing the transgene, promoter, intron (if one is to be used), enhancer, polyA sequence, and optionally a signal sequence or membrane anchoring domain into the embryo. This may be accomplished by cutting the plasmid so as to remove the nucleic acid sequence region containing these elements, and purifying this region using agarose gel electrophoresis or other suitable purification methods .

2. Production of Transσenic Mammals

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 fitness. For example, when transgenic mice are to be produced, lines such as C57/BL6 x DBA2 Fl cross, or FVB lines are often used (obtained commercially from Charles River Labs, Boston, MA) . The line(s) used to practice this invention may themselves be transgenics, and/or may 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 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 injections.

Similarly, the male mammal to be used as a stud will normally be selected by age of sexual maturity, among other criteria.

Administration of hormones or other chemical compounds may be necessary to prepare the female for egg production, mating, and/or reimplantation of embryos. The type of hormones/cofactors and the quantity used, as well as the timing of administration of the hormones 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 (i.e., one that is producing eggs that can be fertilized) is mated with a stud male, and the resulting fertilized embryos are then removed for introduction of the transgene (s) . Alternatively, eggs and sperm may be obtained from suitable females and males and used for in vitro fertilization to produce an embryo suitable for introduction of the transgene.

Normally, fertilized embryos are incubated in suitable media until the pronuclei appear. At about this time, exogenous nucleic acid comprising the transgene of interest is introduced into the female or 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, microinjection, electroporation, or lipofection. Following introduction of the transgene nucleic acid sequence into the embryo, the embryo may be incubated in vitro for varying amounts of time, or reimplanted into the surrogate host, or both. In vitro incubation to maturity is within the scope of this invention. One common method is to incubate the embryos in 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 anesthetized, and the embryos are inserted into the oviduct. The number of embryos implanted into a particular host will vary by species, but will usually be comparable to the number of offspring the species naturally produces .

Transgenic offspring of the surrogate host may be screened for the presence and/or expression of the transgene by any suitable method. Screening is often accomplished by Southern blot or Northern blot analysis, using a probe that is complementary to at least a portion of the transgene. Western blot analysis using an antibody against the protein encoded by the transgene may be employed as an alternative or additional method for screening for the presence of the transgene product. Typically, DNA is prepared from tail tissue (about 1 cm is removed from the tip of the tail) and analyzed by Southern analysis or PCR for the transgene. Alternatively, the tissues or cells believed to express the transgene at the highest levels are tested for the presence and expression of the transgene using Southern analysis or PCR, although any tissues or cell types may be used for this analysis. Alternative or additional methods for evaluating the presence of the transgene include, without limitation, suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular markers or enzyme activities, and the like:. Analysis of the blood may also be useful to detect the presence of the transgene product in 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 transgenic mammals may be obtained by mating the transgenic mammal with a suitable partner, or by in vitro fertilization of eggs and/or sperm obtained from the transgenic mammal. Where mating with a partner is to be performed, the partner may or may not be transgenic and/or a knockout; where it is transgenic, it may contain the same or a different transgene, or both. Alternatively, the partner may be a parental line. Where in vitro fertilization is used, the fertilized embryo may be implanted into a surrogate host or incubated in vitro, or both. Using either method, the progeny may be evaluated for the presence of the transgene using methods described above, or other appropriate methods.

The transgenic mammals of this invention may be used to generate one or more cell lines. Such cell lines have many uses, as for example, to evaluate the 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 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 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 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 several 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 conditions and diseases, and/or (b) screening compounds that have potential as prophylactics or therapeutics. Such uses may be found for (1) conditions caused by inflammation, (2) immune system disorders, (3) epithelial cell repair (skin, lung and/or intestinal epithelia) , (4) hematopoiesis, and/or (5) disorders caused by various physical 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 response to the compound(s) over time. Administration may be by any appropriate means such as, for example. oral administration, or administration by injection, implantation, or transdermal delivery, depending on the chemical nature of the compound being evaluated. In some cases, it may be appropriate to administer the compound in conjunction 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 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 medium in conjunction with other compounds or co-factors that might enhance the efficacy 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 present invention.

EXAMPLES

Example 1: Preparation of a FABP promoter/IL-8 Mouse

A. Preparation of DNA Constructs

The overall cloning strategy used to prepare the FABP promoter-IL-8 transgene construct is depicted 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 tissue. An approximately 1.2 kb fragment between nucleotide positions 1 and 1210 of the rFABP sequence was generated. This sequence, is set forth and numbered according to the Genbank sequence (Accession Number M18080; Sweetser et al . , J. Biol . Chem . , 262:16060-16071 [1987]) . This sequence of 1210 nucleotides is shown in Figure 1. The primers used for amplification of this sequence were complementary to the 5 ' and 3 ' ends of the sequence, and were designed to create an EcoRI restriction site at either end of the rFABP promoter fragment. The amplified fragment was inserted into the EcoRI site of pUC19 (New England Biolabs, Beverly, MA) to generate the plasmid FABPτ_B.

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 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 Kpnl site at either end of the amplified sequence. The primer sequences were:

Primer 1: CGGAATTCCGGAGGTGAAGGACGTCCTTCC (SEQ ID NO: 2)

Primer 2: CGGAATTCCGATTTGTAGGCCTTCAACTCC (SEQ ID NO: 3)

Thirty cycles of amplification were conducted as follows: Denaturation was at 94°C for 30 seconds; annealing was at 54°C for 30 seconds; and extension was at 72°C for 30 seconds.

The PCR product of about 800 base pairs was digested with Kpnl and inserted into Kpnl cut FABP_TB. The resulting vector was designated FABP-Eintron. The promoter-ApoE intron cassette was excised from FABP- Eintron as an EcoRI fragment and cloned into EcoRI cut pBIISK+ (Stratagene Corp., La Jolla, CA) to generate the plasmid pFE-BS. The promoter-intron cassette was subcloned into this vector in both orientations for future use.

The human IL-8 cDNA was obtained by screening 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) . Mononuclear 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., St. Louis, MO) for 19 hours, followed by addition of cycloheximide to a final concentration of 10 ug/ml for an additional 6 hours. Incubation was carried out at 37°C and 5% C0₂.

Total RNA was isolated from activated lymphocytes using the guanidium thiocyanate-CsCl technique (Chirgwin et al., Biochem. , 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 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 was synthesized following the procedure set forth in

Sambrook et al . , supra, followed by methylation with Eco RI and Alu methylases . The technique of Dorssers et al, {Nuc. Acad. Res . , 15: 3629, [1987]) was used to introduce Eco RI and Hind III sites on the 5' and 3* ends of the cDNAs, respectively. After digestion with Eco RI and Hind III restriction enzymes, cDNAs that were larger than 500 base pairs were isolated from an agarose gel by electroelution. The eukaryotic expression vector V19-10 (described above) , was digested with Eco RI and Hind III and was then ligated with the cDNAs. These new plasmids containing cDNA inserts were transfected into competent DH5 alpha cells (GIBCO-BRL, Gaithersburg, MD) . The cDNA library was frozen in aliquots at -80°C after addition of DMSO to 7% (Okayama & Berg, Mol . Cell . Biol . , 2: 161-170, 1982) . A mixed oligonucleotide probe was designed 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:

ATGTCGACMWCSVTGCMCCHRYMYSMYCYA (SEQ ID NO : 4 )

In this sequence , M, W, S, V, R, Y, and H represent degenerate nucleotides . M represents A or C; 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 H represents A or C or T .

Using this probe, a cDNA encoding IL-8 was obtained . The IL-8 cDNA clone was sequenced to confirm that it was homologous with the published sequence (Furutani et al . , Biophys . Biochem . Res . Comm . , 159 : 249- 255 [1989] ) . This IL-8 cDNA was then used as a template to PCR amplify a Spel-NotI fragment of the cDNA. Amplification was accomplished using the following oligonucleotide primers :

Primer 3 : GGACTAGTCCAGAGCACACAAGCTTCTAG (SEQ ID NO : 5)

Primer 4 : ATAAGAATGCGGCCGCTAAACTATTGCATCTGGCAACCC (SEQ ID NO : 6) Thirty cycles of amplification were conducted as follows: Denaturation was at 94°C for 30 seconds; annealing was at 54°C for 30 seconds; and extension was at 72°C for 30 seconds. The amplified fragment was then subcloned into

Spel-NotI cut pIIBS-PA (NS) to produce the plasmid pIL-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 expression vector V19-10 was used as a template for amplification of the SV40 polyA+ signal. This vector, V19-10, was constructed by inserting a 592 base pair Aatll/Clal fragment containing the origin of replication sequence from bacteriophage M13 into the eukaryotic expression vector V19-8 (described in WO 91/05795, published May 2, 1991) . The approximately 242 base pair polyA÷ sequence from VI9-10 was amplified as a Notl- SacII fragment or a Hindlll-Xhol fragment using PCR. The primers used for PCR amplification were:

Not l-SacI I fragment :

Primer 5 : CTCTAGAAAGCTTAATTCAGTC (SEQ ID NO : 7 )

Primer 6 : TCCCCGCGGGGAAGAGCGCAGAGCTCGG (SEQ ID NO : 8 )

Thirty cycles of amplification were conducted as follows : Denaturation was at 94°C for 30 seconds ; annealing was at 56°C for 30 seconds ; and extension was at 72°C for 30 seconds .

Hindlll-Xhol fragment :

Primer 7: CTCTAGAAAGCTTAATTCAGTC (SEQ ID NO: 9)

Primer 8: CTGGATCTCGAGGTACCCGGGGATCATAATC (SEQ ID NO: 10) Thirty cycles of amplification were conducted as follows: Denaturation was at 94°C for 30 seconds; annealing was at 57°C for 30 seconds; and extension was at 72°C for 30 seconds. The PCR fragments were sequenced and showed

100% homology to the template. The fragments were then subcloned into Notl-SacII cut or Hindlll-Xhol 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 * untranslated sequence of the original IL-8 cDNA, was sequence verified and found to be 100% homologous to human IL-8 in the coding region.

To make the final construct for microinjection of mouse embryos, the promoter-intron casette was excised from pFE-BS as a Xhol-Spel fragment and subcloned into Xhol-Spel cut pIL-8PA to generate the plasmid pFE-IL-8 PA, also called FE8.

For microinjection into mouse embryos, the vector was digested with Xhol, Seal and Afllll, to obtain an approximately 3.1 kb Xhol-Afllll insert fragment containing the rFABP promoter, 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 microinjectio .

Pregnant mare's serum ("PMS"), supplying Follicle Stimulating Hormone ("FSH") was administered to female mice of the strain BDF1 (Charles River Labs, Boston, MA) about three days prior to the day of microinjection. PMS (obtained from Sigma Chemicals) was prepared as a 50 I.U./ml solution in Phosphate Buffered Saline and injected intraperitoneally at 0.1 ml (5 I.U.) per animal. Human Chorionic Gonadotropin ("HCG"), supplying Luteinizing Hormone ("LH") was administered 45-48 hours after the PMS injections. HCG was also prepared as a 50 I.U./ml solution in PBS and injected IP (intraperitoneally) at 0.1 ml per animal. Females were placed with stud males of the same strain immediately after HCG injections. 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 cervical dislocation, and both oviducts were rapidly removed and placed in M2 medium (Hogan et ai., eds., Manipulating the Mouse Embryo : A Laboratory Manual, Cold Spring Harbor Laboratory Press, pp 249-257 [1986]) . The oviducts were transferred individually from M2 medium to PBS containing 300 μg/ml hyaluronidase (Sigma Corp., St. Louis, 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 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 equilibrated M16 medium (Hogan et al . , supra) , placed in equilibrated microdrop dishes containing Ml6 medium under paraffin oil and returned to the incubator.

Fertilized single-cell embryos from BDF1 xBDFl-bred mice were selected in Ml6 medium and incubated about 5 hours at 37°C 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 holding pipet, a single embryo was selected and rotated such that the male pronucleus was away from the holding pipet. Approximately 2 to 3 picoliters of solution containing the DNA construct at about 1 microgram per ml was injected into one of the pronuclei, preferably the male pronucleus. Following the injection, the embryos 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 CD1 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 microscope 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. The ovary and oviduct were separated to expose the opening of the oviduct (termed the infindibulum) . Surviving embryos were then removed from the incubator and loaded into the reimplantation pipet. The tip of the pipet was inserted several millimeters into the infindibulum and gentle pressure was used to deliver the embryos into the oviduct. About 10 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 sutured.

B. Identification of Transgenic Mice

Of 56 mice born after embryo injections, 11 contained the IL-8 transgene as assayed by PCR amplification. About 1 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 subjected to PCR analysis using the following primers: Primer 9: GCCTCTAGAAAGAGCTGGGAC (SEQ ID NO: 11)

Primer 10: CGCCGTGTTCCATTTATGAGC (SEQ ID NO: 12)

The PCR amplification procedure was denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 30 seconds. Thirty cycles were performed.

The resultant transgenic mice harboring the transgene in their genome are termed the founder mice.

The founder mice were backcrossed to strain BDFl mice to generate heterozygous Fl transgenic mice.

To evaluate the Fl 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 Fl 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 manufacturer's protocol. Prior to counting, red blood cells were lysed with Quicklyser™ (Toa Medical Electronics Co., LTD, Kobe, Japan), following the manufacturer's protocol. For differential leukocyte analysis, about 3 μl 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 visualizing the cells under a lOOx oil emersion lens on an Olympus CH2 student microscope . Neutrophils were distinguished from lymphocytes, macrophages, eosinophils, and basophils by their multinucleated structures. For all lines reported, at least five 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.

Example 2 : Preparation of a FABP promoter/KGF Mouse

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 1) with the restriction endonucleases Xhol and Spel and isolating the fragment containing the rat FABP promoter-ApoE intron sequence (3' portion of exon 1, full length sequence of intron 1, and 5' portion of exon 2; described in Example 1) . This cassette was then inserted into the vector pIIBS-PA (NS) , which is described in Example 1. The resulting vector, which contains the rat FABP promoter and ApoE sequence upstream of a polylinker and SV40 polyadenylation site was designated pFEPA#3.

The gene encoding human KGF (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: Primer 11 : CAATCTACAATTCACAGA (SEQ ID NO : 13)

Primer 12 : TTAAGTTATTGCCATAGG (SEQ ID NO: 14)

The conditions for PCR were : denaturation at 92°C for 20 seconds ; anneal at 55-40°C for 20 seconds (this consisted of 2 cycles at 55°C, followed by 2 cycles at 45°C, which was followed by 28 cycles at 40°C) ; and extension at 72°C for 30 seconds . Thirty cycles total were performed .

To introduce Hindlll and Bglll restriction sites to the ends of the KGF cDNA, the cDNA was PCR amplified using the following two oligonucleotide primers :

Primer 13: AACAAAGCTTCTACAATTCACAGATAGGA (SEQ ID NO: 15)

Primer 14: AACAAGATCTTAAGTTATTGCCATAGG (SEQ ID NO: 16)

The conditions for PCR were: denaturation at 92°C for 20 seconds; anneal at 45°C for 20 seconds; and elongation at 72°C for 30 seconds. Thirty cycles were performed.

After amplification, the KGF cDNA was purified and digested with Hindlll and Bglll, and then ligated into the vector pCFM3006. This vector was prepared from the vector pCFM836 (described in U.S. Patent No.

4,710,473, issued December 1, 1987) . The two endogenous Ndel restriction sites in pCFM836 were removed by cutting pCFM836 with Ndel, filling in the cut ends of the vector using T4 polymerase, and then re-ligating the vector by blunt end ligation. Next, the DNA sequence between the Aatll and Kpnl 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 base pair position changes are relative to the Bglll site on pFM836 which is position #180) : plasmid bp # bp change

# 428 G/C

# 509 A/T

# 617 insert two G/C bp

# 978 C/G

# 992 A/T

# 1002 C/G

# 1005 T/A

# 1026 T/A # # 1 1004455 T/A

# 1176 T/A

# 1464 T/A

# 2026 bp deletion

# 2186 T/A

## 22447799 T/A

# 2498-2501 £-I£Δ

# 2641-2647 bp deletion

# 3441 A/T

# 3649 T/A

The KGF cDNA in this vector was used as a template for amplification. A 710 base pair Hindlll fragment of KGF was amplified using PCR and the following two oligonucleotide primers:

Primer 15: CGATCGTAAGCTTGGTCAATGACCTAGGAGTAAC (SEQ ID NO: 17)

Primer 16: CGATCGTAAGCTTGCGGATCCTAAGTTATTGCC (SEQ ID NO: 18)

Amplification was conducted for 30 cycles. Denaturation was at 94°C for 30 seconds, annealing was at 58°C for 20 seconds, and elongation was at 72°C for 30 seconds. The amplified fragment was purified by agarose gel electrophoresis, and then was ligated into the vector pFEPA#3 which had been previously digested with Xbal. The ligated vector containing the KGF insert was transformed into E coli DH5 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 alkaline lysis method along with CsCl gradient centrifugation. The purified plasmid was designated FEK.

For microinjection into mouse embryos, the plasmid FEK was digested with Xhol, Seal, and Afllll to obtain a 3.3 kb Xhol-Afllll fragment containing the rFABP promoter, a portion of the ApoE first exon, the ApoE first intron, a portion of the second exon, the human KGF 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 microinjection. Microinjection and implantation into pseudopregnant mice were conducted as described in Example 1. 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 1.

All literature cited herein is specifically incorporated by reference. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Amgen Inc.

(ii) TITLE OF INVENTION: Enhanced Transgene Expression

In Specific Tissues

(iii) NUMBER OF SEQUENCES: 18

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: 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) MEDIUM TYPE: Diskette, 3.5 in., DS, 2.0 Mb

(B) COMPUTER: Apple Macintosh

(C) OPERATING SYSTEM: Macintosh OS 7.0.

(D) SOFTWARE: Microsoft Word Version 5.1a (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: 18-OCT-1993

(C) CLASSIFICATION:

(2) INFORMATION FOR SEQ ID Nθ:l: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1210 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l: GAATTCCTTA ATTTGCATAA TTTACTCACA TTAGTCAAGA ACAAAACATT 50

TAAAAAAATA AAGAGGTAGC TGATATGTAA ATACTGAAAA CACATTCGGT 100

GAAAAGATTC AAACATAAAA AGACTACCTG ATATATAATT ATATTTGTAT 150

GAAATGTCCA TAGAGGTAGA AAGTGGATGG GAACCTAATG GTCTAAAAAT 200

AGAAAATGAG AGCTGGAGAG ATGGCTCAGC AGTTAAAAGC ACTGACTGCT 250

CTTCCAGAGG ACCTGAGTTC AATTCCCAGC AACCACATGG TGGCTCACAA 300

CCATCTGTAA TGGGATCTGA TGCCCTCTTC TGGTGTGTCC AAAGACAGCG 350

GTGGTGTACT CACATACATA AAGTAAAAAA TTCTTTAAAA AAAAAATAGA 400

AAATGAAAAC CCACTGCTTA AAATGTATGC AGTTTCTTTT GGGGATAATG 450

ACACTGTTTC AGAATATGAT AATAGAGATG TTTGGGCAAA TAGTTTATAT 500

ACTAAATTTT AGATTGATTT GATATCAATA AAATAACCTT AGGAACACAG 550

AAAAATTAAA ATACATTTTT TAGTTCCAAT AATCACTTAA ACTTTAACTT 600

TGCAATTGCA CTTTTGTATA GTTTGTTCAA CTCCTGTTAA AGATTCGTGG 650

TATAAGTCTC ACATCCCAGT TGTTTGACGT TTCTTACAAA TGATACATAG 700

TTACATAATT ACATAGTTAC ACCTGCTGTA GAGTGTGCAC TTTGAACCTG 750

TAAATAGAAA AGTATCTTGA GGGTTTTTTC TTCTTGTTGT TGTTTGTCTG 800

TTTGGTTTGG TTTATCGCCT GATTCATTCT TGTTGTTTGT CTTTTGGCTG 850

GAGTGGAACT CCTTATTACA GCAGGCTAGA ATTGTCTGCC TCTGCTTCTG 900

GAGAGCTCGG ATTAAAGGTG GAAGCCATCA CACTTGACCC TAATTCTTGG 950

AATAAAAATG CCTACATGCT GTAGTCGGAG ACAGAGTAGG TATGGTTACC 1000

AAATTTGAAT GCAGTTGAAT CTCAGCAATA GATTCAAAGA AAGCACTAGA 1050

AGAGAAACTA AAGGGGCCTG GCATGCAAAA CTGCCAGGTT ATCTCTTGAA 1100

CTTTGAACTT CCACATCATG GTATGAATTG GTTCGAAGAT AAGAAATAGA 1150

ATAAATTCTC TCTAGTGGAC AGGACCGAAT CTCTGCTTTC CTAGAGGCAC 1200

ACACAGCTGA 1210

(2) INFORMATION FOR SEQ ID Nθ:2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CGGAATTCCG GAGGTGAAGG ACGTCCTTCC 30

(2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CGGAATTCCG ATTTGTAGGC CTTCAACTCC 30

(2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: ATGTCGACMW CSVTGCMCCH RYMYSMYCYA 30

(2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: GGACTAGTCC AGAGCACACA AGCTTCTAG 29

(2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: ATAAGAATGC GGCCGCTAAA CTATTGCATC TGGCAACCC 39

(2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CTCTAGAAAG CTTAATTCAG TC 22

(2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TCCCCGCGGG GAAGAGCGCA GAGCTCGG 28

(2) INFORMATION FOR SEQ ID NO:9: - 4l -

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CTCTAGAAAG CTTAATTCAG TC 22

(2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: CTGGATCTCG AGGTACCCGG GGATCATAAT C 31

(2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll: GCCTCTAGAA AGAGCTGGGA C 21

(2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CGCCGTGTTC CATTTATGAG C 21

(2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: CAATCTACAA TTCACAGA 18

(2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: TTAAGTTATT GCCATAGG 18

(2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: AACAAAGCTT CTACAATTCA CAGATAGGA 29

(2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: AACAAGATCT TAAGTTATTG CCATAGG 27

(2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: CGATCGTAAG CTTGGTCAAT GACCTAGGAG TAAC 34

(2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

CGATCGTAAG CTTGCGGATC CTAAGTTATT GCC 33

Claims

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-α interferon-β, interferon-γ, 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 5¹ 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 3¹ 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 3¹ end to the 5¹ 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.