MXPA98010665A - Vectors and methods for synthesis specific to protein tissue ingengos de gallinas transgeni - Google Patents

Abstract

Vectors and methods are provided for introducing genetic material into cells of a chicken or other avian species. More particularly, vectors and methods are provided to transfer a transgene to an embryonic chicken cell, to create a transgenic hen where the transgene is expressed in the hen's oviduct and the transgene product is secreted in the chicken's eggs and / or those of your offspring. In a preferred embodiment, the product of the transgene is secreted in the egg blan

Description

VECTORS AND METHODS FOR SYNTHESIS SPECIFIC TO PROTEIN TISSUE IN EGGS OF TRANSGENIC HENS DESCRIPTION OF THE INVENTION This application is a continuation in part of U.S. Application Serial No. 60 / 019,641, entitled "Vector For Expression of Proteins Into Eggs of Transgenic Hens," filed June 12, 1996. The present invention relates generally to vectors and methods for introducing genetic material into an embryo of a chicken and other species of birds and, more particularly, to vectors and methods for transferring a gene of interest to an embryonic chicken cell, to thereby create a chicken transgenic that has the gene of interest expressed in the oviduct of the hen and the gene product secreted in the eggs of the hen and / or those of their offspring. Since the development of recombinant DNA technology, some twenty-five years ago, the prospect of producing proteins on a large scale, rather than extracting them from tissue where they are naturally expressed, has become a reality. In particular, in the last two decades, progress in the development of expression vectors has led to the production of thousands of recombinant proteins on a laboratory scale. The production of commercial quantities of recombinant proteins often requires difficult and expensive expansion procedures, but it has also been successful. In addition, transgenic animals including mice, rabbits, pigs, sheep, goats and cows have been engineered to produce human pharmaceuticals in their tissues or secretions. Houdebine, L.M., J. Biotechnology 34: 269-287 (1994). Although the white egg is thought to be an excellent host for production of recombinant protein, the preparation of transgenic birds has proven to be technically difficult due in large part to problems involved in the handling of chicken embryos. When oviposition occurs, the embryo has already reached a stage that corresponds to a late blastula or mammalian early gastrula. Genetic manipulation of the embryo during early development requires reintroduction to the female or in vitro culture, both difficult procedures. Houdebine, L.M., J. Biotechnology 34: 269-287 (1994). Despite these difficulties, transgenic chickens that are resistant to avian leukosis infection (Crittenden and Salter, "Transgenic Livestock Models in Medicine Adn Agriculture" page 73-87 (Wiley-Liss (1990)) or have high levels have been produced. of circulating growth hormone Bosselman, RA, et al., Science 243: 533-535 (1989) Four general methods for generating transgenic birds have been reported, one method involves excision of an oviduct-developed egg, DNA microinjection near of the blastoderm, and in vitro culture of the manipulated embryo in solution and substitute husks Love, J., et al., Biotechnology 12: 60-63 (1994). A second method requires the cultivation and transfection of primogenial germ cells, with subsequent transplantation in an irradiated recipient near the same stage of development as the donor. Carsience et al., Development 117: 669-675 (1993); Etches et al., Poultry Science 72: 882-889 (1993). Although technically they are in high demand, these two procedures are attractive since large pieces of DNA can be transferred. A third method involves blind injection of replication competent retroviruses with a needle near the blastoderm of a recently laid egg. Petropoulos, C.J., et al., J. Virol. 65: 3728-3737 (1991). Although this method is the simplest, it is also limited since the DNA to be transferred must be approximately 2 kb or less in size and, the method results in viraemic hens which release infective recombinant retrovirus. Petropoulos, C.J., et al., J. Virol. 66 (6): 3391-3397 (1992). The fourth method involves a retroviral vector system defective in replication (see, for example, U.S. Patent Nos. 5,162,215 and 4,650,764, incorporated herein by reference). One of these systems (Watanabe and Temin, Mol. Cell, Biol. 3 (12): 2241-2249 (1983)) has been derived from type A reticuloendotheliosis virus (REV-A). Sevoian et al., Avian Dis. 8: 336-347 (1964). Retroviral vectors defective in replication derived from the REV-A virus are based on the C3 helper cell line (Watanabe and Temin, Mol.Cell. Biol. 3 (12): 2241-2249 (1983)) which contains the components of a defective auxiliary provirus in packaging. The derivation of the C3 helper line and several defective retroviral vectors in replication has been described in detail in U.S. Patent No. 4,650,764 and Watanabe and Temin, Mol. Cell. Biol. 3 (12): 2241-2249 (1983). This method is more technically demanded than the replication competition technique since the blastoderm must be exposed, and microinjection equipment must be used. Bosselman, R.A. , et al., Science 243: 533-535 (1989). However, it results in transgenic hens free of replication competition retroviruses, and can transfer DNA as long as 8 kb in size. Tissue-specific expression of a foreign gene is achieved in a transgenic chicken using the replication-competent retrovirus technique. Petropoulos, C.J. et al., J. Virol. 66 (6): 3391-3397 (1992). A replication-competent retrovirus is used to deliver the chloramphenicol acetyl transferase reporter gene (CAT), driven by a muscle-specific promoter, action a, for skeletal muscle. The tissue-specific expression of a recombinant protein in the egg of a transgenic bird has not yet been successful.

In this way it is desirable to provide a vehicle and method for transferring a gene to an embryonic chicken cell (or other avian species) to thereby create a transgenic hen wherein the gene is expressed in a tissue-specific form. It may also be desirable to provide a vehicle and method for transferring a gene to an embryonic chicken cell, wherein the gene is expressed in the oviduct of the hen and the secretion of the gene product is in the eggs of the hen. It may be desirable to provide a vehicle and method for transferring a gene to an embryonic chicken cell, wherein the gene is expressed in the chicken's oviduct and the secretion of the gene product is in the chicken's eggs without compromising the health of the chicken. the hen and the health of other birds in contact with her. Vectors and methods are provided for introducing genetic material into the cells of a chicken or other bird species. More particularly, vectors and methods are provided to transfer a transgene to an embryonic chicken cell, to thereby create a transgenic hen where the transgene is expressed in the oviduct of the hen and the transgene product is secreted in the eggs of the hen and / or those of their offspring. In a preferred embodiment, the product of the transgene is secreted in the white egg. In one embodiment, the vector comprises a portion of a retroviral genome, capable of transfecting a cell and incapable of replication, ie, a retroviral vector defective in replication. The vector further comprises a transgene, operably linked to appropriate control elements such that the transgene can be expressed in a tissue-specific manner. In one embodiment, the control elements include an improved promoter that directs expression of the transgene in the oviduct, a 5"untranslated region for the structural gene (coding region) of appropriate length and sequence to promote efficient translation, and a signal sequence that directs the secretion of the transgene product in the white egg In this embodiment, the promoter can be chosen, without limitation, from the group consisting of ovalbumin, lysozyme, conalbumin and ovomucoid promoters, and combinations thereof. another modality, the control sequences include a promoter that directs the expression of the transgene in the liver and a signal sequence that directs the uptake and secretion of the transgene product in the egg yolk. In this embodiment, the promoter can be chosen, without limitation, from the group consisting of vitellogenin and apolipoprotein promoters, combinations thereof. The vectors of the present invention can be used in the production of transgenic birds, particularly chickens, by methods known to those skilled in the art, such as the four methods described above (see, Background Of The Invention). For example, as described in U.S. Patent No. 5,162,215, incorporated herein by reference, vectors can be used to introduce a nucleic acid sequence, e.g., a gene, into germ cells and germ cells. of an embryo of a chicken. In one embodiment, the vector is microinjected into a recently laid chicken egg. In close proximity to (for example directly below) blastoderm. The egg is then sealed and incubated until the chicken has hatched from the egg. The transgenic chicken is then tested for transgene expression and if it is positive and the chicken is female (chicken), the chicken eggs are harvested and the protein is isolated by methods known to those skilled in the art. If the chicken is male (rooster), it can be reproduced to produce a female transgenic chicken whose eggs can then be harvested. In this way they are provided, birds and transgenic eggs, as well as methods to make birds and transgenic eggs. Other features and advantages of the present invention will become apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS The various advantages of the present invention will become apparent to one skilled in the art by reading the following specification and sub-annex claims and for reference the following drawings in which: Figure 1 is a schematic illustrating the production of the vectors of the present invention and methods for using same to produce transgenic chickens; Figure 2 is a schematic illustrating a preferred vector of the present invention; Figure 3 is a schematic illustrating the construction of the retroviral and expression vectors of the present invention; Figure 4 is a schematic illustrating the construction of intermediary # 1, pOVSV; Figure 5 is a schematic illustrating the construction of intermediate # 2, pSigl (SEQ ID NO 2-7); Figure 6 is a schematic illustrating the construction of intermediate # 3, pSigPCR (SEQ ID NO: 8-9); Figure 7 is a schematic illustrating the construction of intermediate # 4, pUTR (SEQ ID NO: 10-13); Figure 8 is a schematic illustrating the construction of intermediate # 5, pUTRAN (SEQ ID NO: 14-15); Figure 9 is a schematic showing the construction of intermediate # 6, pERE 1; Figure 10 is a schematic showing the construction of intermediate # 7, pERE (note in Figure 10 that the arrows are for the orientation of the ERE sequence within the oligonucleotides, not the oligonucleotide itself) (SEQ ID NO. 25); Figure 11 is a schematic illustrating the modified proviral vector (SEQ ID NO: 26-27); Figure 12 is a schematic illustrating the modification of the 3"end of the hygromycin B phosphotransferase gene (SEQ ID NO: 30-31), and Figure 13 is a schematic showing the modification of the N terminus of the hygromycin B gene. phosphotransferase (SEQ ID NO: 28-29): Vectors and methods are provided for introducing genetic material into the cells of a chicken or other avian species, More particularly, vectors and methods are provided for transferring a transgene to embryonic chicken cells, for thus creating a transgenic hen where the transgene is expressed in the oviduct of the hen and the transgene product is secreted in the chicken eggs and / or those of their offspring Figure 1 is a schematic illustrating the methods of the present invention, including production of the vector and use to produce a transgenic chicken In one embodiment, the vector comprises a portion of a retroviral genome, capable of transfecting a cell a and incapable of replication, that is, a retroviral vector defective in replication. Retroviral vectors defective in replication derived from REV-A viruses are preferred. In addition, the vector comprises a gene of interest also referred to herein as a transgene, operably linked to appropriate control elements such that the transgene product can be synthesized in a tissue-specific manner. In Figure 2 a schematic of a preferred expression factor of the present invention is indicated. It will be appreciated that the 3 kb gene of β-galactosidase shown in Figure 2 is simply a reporter gene and is replaced with any transgene or fragment thereof. For example, a gene which encodes a blood coagulation protein such as fVIII may be employed. The product of the transgene or protein is secreted in the egg and is then isolated. Once purified, the protein can be used in applications such as in the treatment of hemophilia. Other preferred genes include, without limitation, genes encoding blood proteins including human serum albumin and 1-antitrypsin, hematopoietic growth factors including erythropoietin, and lymphopoietic growth factors such as granulocyte colony stimulating factors. Genes coding for industrial proteins such as -amylase and glucose isomerase can also be employed. On the other hand, genes encoding antibodies and immunoreactive portions thereof can also be included in the vectors of the present invention (see, for example, Lilley, et al., J. I munol, Meth. 171: 211-226 ( 1994) and Davis et al., Biotechnol., 9: 165-169 (1991), incorporated herein by reference). The gene, or gene fragment, to be transferred can be produced and purified by any methods well known in the art. Thus, a gene can be produced synthetically, or by treating mRNA derived from the transcription of the reverse transcriptase gene to produce a cDNA version of the gene, or by direct isolation of the gene from a genomic bank or from other sources. Control elements which flank the transgene include promoters and amplifiers, UTR and signal sequences, which allow tissue-specific expression of the transgene. In one embodiment, the promoter directs the expression of the transgene in the oviduct of the transgenic bird. A preferred promoter of the present invention is chosen from the group consisting of ovalbumin, lysozyme, conalbumin and ovomucoid promoters, and combinations thereof. The signal sequences included in the vector direct the secretion of the transgene product in the white egg. In an alternative embodiment, the promoter drives the expression of the transgene in the liver and signal sequences included in the vector direct the secretion and uptake of the transgene product in the egg yolk. In this embodiment, the promoter of the group consisting of of vitellogenin and apolipoprotein A promoters, and combinations thereof. Preferred amplifiers are viral amplifiers including, but not limited to, the SV40 amplifier, or portion thereof. Amplifiers of lysozyme in addition to synthetic DNA may also be employed although to bind to transcription factors, such as spheroid hormone response element, for example the random EREs described herein. In one embodiment of the present invention, shown in Figure 2, control elements flanking the gene of interest include the SV40 amplifier, three random estrogen response elements (ERE), 1.3 kb of the ovalbumin promoter (flanking 5") , 77 bp of the 5"untranslated region (UTR), the N-terminal signal peptide sequence of the chicken lysozyme gene, and the polyadenylation and termination signals of the small SV40 T antigen. SEQ ID NO: 1 indicates the nucleotide sequence of the preferred construct. In a preferred embodiment of the present invention, this construct is contained in a 5 kb Xba I fragment which is inserted into a retroviral vector defective in transgenesis replication. The preferred proviral vector is a derivative of the plasmid pSW272. Emerman, M., et al., Cell 39: 459-467 (1984); U.S. Patent No. 4,650,764. As described in U.S. Patent No. 4,650,764, incorporated herein by reference, cell lines have been constructed to complement these vectors and produce viral proteins necessary to package retroviral vectors defective in replication. The packaged vector can infect a cell once, but is itself incapable of infecting in subsequent turns. The vectors of the present invention are particularly useful in the production of transgenic birds, particularly chickens, by methods known to those skilled in the art. For example, as described in U.S. Patent No. 5,162,215, incorporated herein by reference, vectors can be used to introduce a nucleic acid sequence, e.g., a gene, into cells of an embryo of a chicken. In one embodiment, the vector is microinjected into a recently laid-up chicken egg in stage X (generally not more than seven days, not incubated), in close proximity to, for example, directly below, the blastoderm. More specifically, an opening of approximately 5 mm in diameter is made on the egg side, usually by the use of a fixed drilling tool with an abrasive rotating tip which can pierce a hole in the egg shell without damaging the membrane underlying the shell. The membrane is then cut by the use of an 18-gauge scalpel or needle and hand clamps, so that a portion of the shell and membrane is removed and the embryo is exposed. The embryo is visualized with the naked eye or with an optical dissection microscope with a 6X-60X amplification. A solution, usually tissue culture medium, containing the vector of the present invention, is microinjected in an area below and around the blastoderm, using a micromanipulator and a needle of very small diameter, preferably glass, external diameter of 40-60. μM at the tip, external diameter of 1 mm along its length. The volume of solution for microinjection is preferably 5-20 μl. After the microinjection, the egg is sealed with a shell membrane and a sealing material, preferably rubber or paraffin. The sealed egg is then incubated at approximately 38 ° C (99.5 ° F) for several periods of time up to and including hatching time to allow the embryo to grow and develop. The DNA of embryos and newly hatched chickens is tested for the presence of microinjected vector sequences. The presence of the inserted sequences is detected by means known in the art and appropriate detection of the specific gene or, if desirable, the product of the gene if the gene or product of the gene, ie, proteina, is present, the eggs of the gene are harvested. the transgenic chickens and the protein is isolated. In another embodiment, the vector or transfected cells that produce the transgene-containing virus are injected into oocytes developed in vivo, for example, as described in Shuman and Shoffner, Poultry Science 65: 1437-1444 (1986), incorporated in the present for reference. The same incubation, hatching, etc. stages are followed. As referred to herein, the term "gene" or "transgene" means a nucleic acid, whether found naturally or synthetically, which encodes a protein product. The term "nucleic acid" is proposed to mean natural and / or linear synthetic, circular and sequential arrays of nucleotides and nucleosides, for example cDNA, genomic DNA (gDNA), mRNA, and RNA, oligonucleotides, oligonucleosides, and derivatives thereof . The term "operatively linked" is proposed to mean joined in a form which allows transcription of the transgene. The term "coding" is proposed to mean that the subject nucleic acid can be transcribed and translated into either the desired polypeptide or the subject protein in an appropriate expression system, for example, when the subject nucleic acid is linked to appropriate control sequences such as promoter and amplifier elements in a suitable vector (e.g., an expression vector) and when the vector is introduced into an appropriate system or cell. As used herein, "polypeptide" refers to an amino acid sequence which comprises both full length protein and fragments thereof. The term "replication defective retroviral vector" refers to a vector comprising a portion of a retroviral genome capable of infecting a cell but incapable of unrestricted replication, i.e., multiple turns of infection, usually due to mutations or deletion in the virus genome. The term "replication-defective vector derived from REV" refers to a viral vector of reticuloendotheliosis that is incapable of unrestricted replication. The term "avian species" includes, without limitation, chickens, quail, turkeys, ducks and other winged. The term "hen" includes all females of the avian species. A "transgenic bird" generally refers to a bird that has had heterologous DNA sequence, or one or more additional DNA sequences normally endogenous to the bird (collectively referred to herein as "transgenes") chromosomally integrated into the bird's germ cells . As a result of such transfer and integration, the transferred sequence can be transmitted through germ cells to the offspring of a transgenic bird. The transgenic bird (including its progeny) will also have the transgene fortuitously integrated into the chromosomes of somatic cells. In order to more fully demonstrate the advantages arising from the present invention, the following examples are indicated. It is understood that the following is for example form only and is not proposed as a limitation on the scope of the invention. SPECIFIC EXAMPLE 1 - CONSTRUCTION OF THE VECTOR Discussion Promoter. Protein ovalbumin is the most abundant protein in the white egg. The ovalbumin is synthesized in the tubular glandular cells of the oviduct and secreted directly into the lumen, where it joins the formed egg. The ovalbumin promoter is a well characterized and complex promoter. Houdebine, L.M., J. Biotech 34: 269-287 (1994). The ovalbumin promoter is regulated by all known classes of steroid hormones (Gaub, MP, et al., Cell 63: 1267-1276 (1990)), and it is thought that at least eight different regulatory proteins or protein groups they are linked to a region of extension 1.1 kb 5 'to the top site.

These proteins include the protein binding complex TATA (TFIID), the estrogen receptor, activator protein 1 (AP-1), which includes the fos and jun gene products and relative peptides (Currant, T., et al., Cell 55: 395-397 (1988)), the upstream promoter transcription factor of ovalbumin chicken (COUP-TF) (Wang, L., et al., Nature 340: 163-166 (1989)) and an associated protein S300-II (Sagami, I., et al., Mol.Cell. Biol. 6 (12): 4259-4267 (1986)), a nuclear protein similar to NF-kB (Schweers, L., et al., J. Biol. Chem. 266 (16): 10490-10497 (1991)), and a nuclear factor I (NF-I) homolog. Bradshaw, M.S., et al., J. Biol. Chem. 263 (17): 8485-8490 (1988). The cis action sequences for these interactions are included in the 1.3 kb fragment used as the preferred promoter in the present invention. Although the natural ovalbumin expression system is mimicked as closely as possible in the vectors and methods of the present invention, the 5 'regulatory region of ovalbumin expands by some 8 kb (Gaub, MP et al., Cell 63: 1267-1276 (1990)), which, along with the other elements downstream (LeMeur, MA, et al., EMBO Journal 3 (12): 2779-2786 (1994)), is very large for a defective retroviral vector in replication. Emerman, M., et al., Cell 39: 459-467 (1984). In this way, the 1.3 kb fragment is used. However, it will be appreciated by those skilled in the art, that the ovalbumin promoter can include any portion of the ovalbumin transcription unit capable of triggering the expression of a transgene in the oviduct. On the other hand, although the ovalbumin promoter is discussed in detail herein, it will be appreciated that other promoters that trigger expression may be employed in cells that generate the white egg, including but not limited to lysozyme, conanbumen and ovomucoid promoters, and combinations thereof. In an alternative embodiment, a promoter that activates the expression of the transgene in the liver, such as the vitellogenin or apolipoprotein A promoter, and combinations thereof, is employed. Although the protein vitellogenin and apolipoprotein A in the yolk are very abundant, they are synthetized in the liver and are then transported to the yolk through the blood. This is deposited in the yolk by means of a specific receptor which recognizes a proximal N fragment of the vitellogenin precursor. Thus, the vectors of the present invention, when containing the vitellogenin or apolipoprotein A promoters (or combinations thereof), also contain a separate signal sequence or sequences that direct the secretion and uptake of the protein in the yolk. Although an intermediate stage of blood transport is required, this type of vector is particularly useful for production of the antibody or compounds found in the blood of other species. Amplifier. The SV40 amplifier has previously been used to increase expression from the ovalbumin promoter. Dierich, A., et al., EMBO Hournal 6 (8): 2305-2312 (1987). AP-2 has been shown to act in the proximal portion of the ovalbumin promoter, and the SV40 amplifier can increase the local concentration of the AP-1 complex or some of its components. Currant, T., et al., Cell 55: 395-397 (1988). There are other control elements found in flanking ovalbumin 5"which are not included in the 1.3 kb ovalbumin promoter Kaye et al., EMBO Journal 5 (2): 277-285 (1986), discover four hypersensitive sites Hormone-dependent DNase in the 5"flanking of the ovalbumin chromatid which correlates with expression of the ovalbumin gene. Two sites are contained within the preferred promoter used herein, and the other two rest 3.3 kb and 6 kb 5"to the top site (sites III and IV respectively). site III, at -3.3 kb, is contained in a Pst I-Xba 675 bp fragment from about 3.7 kb to 3.1 5 'to the top site. Within this fragment are four medium palindromic estrogen response (ERE) elements which increase the expression of the ovalbumin promoter in a synergistic manner. Kato, S., et al., Cell 68: 731-742 (1992). The average EREs are spaced more than 100 base pairs apart. However, fusion and elimination studies have demonstrated both the functionality and necessity of these elements to confer the estrogen response to a truncated ovalbumin promoter. Kato, S., et al., Cell 68: 731-742 (1992). It is thought that several loosely linked estrogen receptors interact synergistically at this locus to result in more stable receptor DNA complexes, which then either destabilize the helix, or increase the local concentration of transcription factors in the vicinity of the promoter. This fragment of region III is not included in the preferred vector of the present invention, but instead is replaced by a synthetic oligonucleotide containing a total palindromic ERE adjacent to and 5"to an individual ERE.The estrogen receptor is bound to ERE palindromic as a dimer with much higher affinity than a single individual site The random arrangement of palindromic ERE and an individual ERE separated by seven base pairs adds additional stability Klein-Hitapaß, L., et al., J. Mol. Biol. 201-537-544 (1988) It is thought that this oligonucleotide functionality replaces the hypersensitive site -3.3 kb in vivo.The random EREs are likely to have a positive effect on gene expression. expression in estrogen response cells, and with imperfect ERE-containing promoters Tsai, SY et al., Cell 57: 443-448 (1989); Ponglikitmongkol, M., et al., EMBO Journal 9 (7): 2221-2231 (1990). There are imperfect EREs in the ovalbumin promoter and synergism is likely to occur between synthetic perfect EREs and natural EREs. The hypersensitive hormone-dependent DNase I site at -6 kb is contained within 1.2 kb. Fusion studies with this DNA fragment show no evidence of improved estrogen response of the ovalbumin promoter. Kato, S., et al., Cell 68: 731-742 (1992). For this reason, no part or analogue is included in the vector shown in Figure 2. Previous investigators have demonstrated an absolute requirement for an intracellular phosphorylation cascade; via somatomedin, insulin, or cAMP for induction of ovalbumin gene in response to estrogen. Evans, M.I., et al., Cell 25: 187-193 (1981); Evans, M.I., et al., Endocrinology 115 (1): 368-377 (1984). Although these studies are more than ten years old and the cascade mechanisms of the second intracellular messenger are now understood in greater detail, the exact mechanism with respect to the cis-specific action sequences in the ovalbumin promoter has not been definitively demonstrated. It is not reasonable to suggest, however, that the mechanism involves binding of AP-1, the cis-acting sequence of which is included in both the preferred ovalbumin promoter and the SV40 amplifier. Curran, T., et al., Cell 55: 395-397 (1988). Untranslated region 5 'the untranslated region 5' (UTR) is that of the ovalbumin wave RNA. The ovalbumin gene contains a 5"guiding exon that is assembled to the first coding exon to generate an untranslated region of 65 bases in length O'Hare, K., et al., Nucleic Acids Rsearch 7 (2): 321-334 (1979). The vector UTR sequence is copied almost exactly to the cDNA to produce 5 'UTR which closely resembles that of the ovalbumin RNA. The only difference is a base mutation near the 5 'end which is necessary for construction, and an additional 3' linker, resulting in UTR of 77 bp in length. A guideline of 77 bases in length is more consistent with the Kozak study which suggests that a minimum of 77 bases is required for maximum translation efficiency (Koxzak, M., et al., J. Cell Biol. 115 (4): 887-903 (1991)). However, it can be used any URT with a functional sequence around the start codon. Signal sequence The signal peptide is responsible for transporting the protein out of the cell, and the signal peptide sequence theory is well developed, von Heijne, G., Eur. J. Biochem. 133: 17-21 (1983); von Heijne, G., J. Mol. Biol. 173: 243-251 (1984); and von Heijne, G., J. Mol. Bíol. 184: 99-105 (1985). In most secreted proteins, the sequence is at the N terminus of the nascent protein and is cleaved during synthesis and translocation in the endoplasmic reticulum. In the case of ovalbumin, however, the sequence is internal to the protein and is not cleaved (Robinson, A., et al., FEBS 203 (2): 243-246 (1986)), thus taking it to be inappropriate for use in an expression vector. The white egg lysozyme signal sequence in the vectors of the present invention is used as a translocation signal since it is a N-terminal cleaved sequence, this functions in vivo in the chicken oviduct, and will release a protein with an N-terminus. native in Saccharomyces. Jigami, Y., et al., Gene 43: 273-279 (1986). However, it will be appreciated by those skilled in the art that any signal can be used. Gene. The β-galactosidase gene is used in the vector indicated in Figure 2 for two reasons. First, at 3 kb, this is the largest of the available reporter genes; many genes that code for commercially valuable proteins are much smaller than this. In this way, if this system can express ß-galactosidase in the egg, then other genes will be expressed in a similar way. Second, the expression of β-galactosidase can be easily assayed, which facilitates the examination of eggs produced from transgenic hens of the present invention. It will be appreciated that any transgenes or fragments thereof can be employed. 3 'control. Since the transgenic vector of the present invention is a retrovirus, the genome is RNA and, a transcription termination signal in the orientation of genome synthesis can prematurely stop the synthesis and result in low retrovirus titers. Bradyopadhy, P.K., et al., Mol. Cell Biol. 4 (4): 749-754 (1984). A transcription termination signal should not alter the synthesis of the genome if it is placed in the opposite orientation, however, but it may not be beneficial in the amplifying effect of a proximal LTR on the retroviral vector. Therefore, both orotations of the expression vector with respect to the retroviral vector are constructed. Standard stop codons and the proven polyadenylation signal of the small SV40 T antigen are included in 3 'of the structural gene. Materials and methods Introduction. The β-galactosidase gene together with the transcription termination signals and the polyadenylation signal of the SV40 small T antigen are contained in a 3.5 kb I-Xba I Cia fragment of the pSVß-galactosidase expression vector, sold by Promega Inc. The ovalbumin promoter is contained in a 1.7 kb Ri Eco Pst I fragment of plasmid pOVl .7 (sequence in Helig, R., et al., J. Mol 156: 1-19 (1982). of Genbank # J00895 M24999). The SV40 amplifier is contained in a 247 bp Neo I-Eoo Rl fragment of the plasmid pCAT amplifier, sold by Promega Ine. All the other DNAs in the construct are synthesized de novo. Figure 3 is a schematic illustrating the construction of retroviral and expression vectors. Construction of the. broker # 1; pOVSV. Plasmid pOVl .6 contains a Hind III site in the first intron of the ovalbumin gene, and a Pst I 1.37 kb site 5"to the top site (see Figure 4) This 1.6 kb Pst I Hind III fragment of pOV1.7 it is linked to Hind III and Nsi I sites of pSVß-galactosidase, (Nsi I has ends compatible with Pst I), resulting in a plasmid called pOVSV, shown in Figure 4. POVSV is the first of 8 intermediaries generated to build the version more complex of the vector Construction of intermediate # 2, pSogl A synthetic linker containing the nucleotide sequence encoding the chicken lysozyme signal peptide is inserted into the Ssp Bl and Co. I sites of pOVSV as shown in Figure 5. The resulting plasmid is called pSigl The nucleotide sequence in Figure 5 is included, together with the amino acid sequence of the signal peptide and the start codon.

Construction of intermediary # 3; pSigPCR. Plasmid pSig I contains undesirable deletions at the 3 'end of the ovalbumin promoter and at the 5"end of the β-galactosidase gene, the β-galactosidase gene is subtracted using PCR, a 3" promoter is used which hybridizes to the β-galactosidase gene. from 35 bp to a unique Saci I site within the gene. The sequence and the design of the PCR are shown in Figure 6. The promoter 5"hybridizes to the 5" end of the β-galactosidase gene and contains 5 'of 17 protruding bases containing a single Csp45 site and 8 nucleotides 5. "Digestion with Csp 45 I generates a compatible end with the Cia digestion. I. CPR is performed for 30 cycles, and the products are digested with Sac I and Csp 45 and then purified on a low melting gel.This 1.9 kb fragment is ligated at the unique Cía I and Sac I sites of pSig I, restoring the β-galactosidase gene and placing it directly in 3"a and in structure with the signal sequence codons (see Figure 6). This plasmid is called pSigPCR, and is verified by digestion with Pvu I, and subsequent sequence analysis. Construction of intermediary # 4; pUTR. A synthetic oligonucleotide encoding 5"UTR of the ovalbumin is ligated into the Bgl II Ssp Bl sites of pSigPCR This oligonucleotide also contains an Acc 65 I site near its 5" end (centered around the stop site) to allow restoration of the promoter in the following stages (see Figure 7). Appropriate constructions are verified by digestion with Kpn I. This plasmid is called pUTR, and contains all the necessary elements 3 'for the top site Construction of the intermediate # 5; pUTRAN. The promoter is restored by ligating a 1.4 bp Neo I partial Ssp Bl restriction fragment containing the total intact pOVSV promoter at the Neo I and Acc 65 I sites, shown in Figure 8. Appropriate recombinants are verified by double digestion with Bgl II Ssp Bl. This plasmid, pUTRAN, has a 1.3 kb ovalbumin promoter that drives all the downstream elements necessary for construction. Construction of intermediary # 6; pERE 1. The elements of response to random estrogen (ERE) are contained in a synthetic oligonucleotide. Since the investigated repeats contained within the EREs form circuit structures from which they prevent annealing in a double-stranded structure, the oligonucleotide is inserted in two stages. The first oligoiucleotide contains two EREs in the same orientation, separated by unique Hind III and Spe I sites. This oligonucleotide is ligated into the Nsi I and Neo I sites of pUTRAN, which forms plasmid pERE 1. pERE 1 also contains Acc III and Neo I sites useful for insertion of the SV40 amplifier, and a terminal Xba I site to allow the insertion of Subsequent constructions in the Xba I site only of the retroviral vector. The appropriate recombinants are verified by Xba I digestion and sequence analysis. Construction of the middleman # 7, pERE. Total palindromic ERE is created by ligation of a synthetic oligonucleotide containing the 3 'mid site at the unique Hind II and Spe I sites of pERE. The resulting plasmid, pERE, contains a total palindromic ERE and a single middle ERE site spaced 7 base pairs (see Figures 9 and 10). Appropriate recombinants are verified by double digestion of Hind III Bgl II, since the ligation of the second oligonucleotide removes the unique Hind III site. Construction of the middleman # 8, pUCERE. The pERE plasmid contains all the elements of the expression vector except the SV40 amplifier. The SV40 amplifier is contained in a 247 bp Eco Rl Neo I fragment of the plasmid pCAT amplifier, available from Promega, pERE contains 3 Eco Rl sites and 2 Neo I sites, necessitating subcloning into a vector which lacks these sites. Plasmid pUC18 contains only one Eco Rl site and lacks a Neo I site as well. PUC18 is digested with Eco Rl and Bam Hl (both at the multiple cloning site), in blunt tip with klenow polymerase and self-ligating. Appropriate eliminations are verified by double digestion with Eco Rl - Sea I. The modified vector is called pUC? BE, and contains a unique Xba I site useful in subcloning the construct. Subsequently, the 5 kb Xba I fragment of pERE, which contains the constructs, is ligated into the modified vector at that site. This plasmid is called pUCERE. Construction of pWMO. PUCERE contains the Neo I and Eco Rl 5 'sites at the ERE, and 3' for the Xba I site necessary to subclone on the retroviral vector. It also contains an extra Eco Rl site within the β-galactosidase gene, which needs a parical digestion strategy. PUCERE is partially digested with Eco Rl, and the linear band isolated. This DNA is digested with Neo I, and the 8 kb fragment is recovered from the low melting gel. The Eco Rl-Neo I fragment of 247 bp of the pCAT amplifier is isolated by standard means, and ligated to the pUCERE preparation. The appropriate recombinants are verified by double digestion of Xba I-Bgl II. This plasmid is called pWVO, and it contains all the transgene elements in a 5kb Xba I fragment. Construction of pBCWM. pWMO and pSW272 both confer resistance to ampicillin to their hosts. To reduce the level of plasmid of origin when subcloned in ampicillin-resistant REV vectors, the 5 kb insert of pWMO is cloned into the unique Xba I site of pBCSK +, purchased from Stratagene (La Jolla, CA), which confers resistance to chloramphenicol for its host. PWMO is digested with Xba I and the 5 kb fragment is isolated from a low melting gel. PBCSK is digested with Xba I, dephosphorylated, and purified on agarose. The vector and insert fragments are ligated with each other, and appropriate recombinants are verified by digestion with Xba I in grown cultures of colonies recovered from plates with LB chloramphenicol (34 μg / ml). This plasmid contains the total expression vector in an origin of resistance to chloramphenicol, ready for insertion in the retroviral vector defective in replication. EXAMPLE 2 SPECIFIC - DESIGN AND CONSTRUCTION OF THE RETROVIRAL VECTOR Design of the vector, retroviral Plasmid pSW272 (Emerman and Temin, Cell 39: 459-467 (1984)) contains a spleen necrosis virus (VNB) deletion mutant, now the reticuloendotheliosis virus (REV). The provirus within the plasmid comprises the LTRs, the packaging sequence and the thymidine kinase gene and its promoter as a selectable marker for viral titer determination. There is a unique Xba I site for the thymidine kinase promoter. In a previous study, the neomycin phosphotransferase gene has been inserted in this vicinity (in a Hind III site) resulting in a second construct pMElll (Emerman and Temin, Cell 39: 459-467 (1984)) which is used successfully to generate transgenic chickens (Bosselman, et al., Science 243: 533-535 (1989)). In the same study, the gene encoding chicken growth hormone is cloned into pSW272, and resulting transgenic chickens have significantly higher levels of circulating growth hormone than non-transgenic controls. PSW272 is modified to better serve as a vehicle for the expression vector. Goals to modify the retroviral architecture include: replacing the thymidine kinase gene with the gene that confers hygromycin B resistance (this eliminates the need for cotransfection, however, it also requires remodeling the ends of the hygromycin gene); Removal of the 5"promoter that drives the hygromycin gene, and its polyadenylation signal (this will provide a more stable architecture), and provide an Xha I 3" site to the hygromycin gene for insertion of the expression vector. The pSW272 is reconstructed in three phases. The first phase is the elimination of the thymidine kinase promoter from the herpes virus and structural gene, and replacement with a synthetic linker. This linker contains sites necessary for subsequent manipulations, including a unique Xba I site at the 3"end which allows the insertion of the 5 kb expression construct contained in pBCWM The second phase is the introduction of linkers to the 5 'ends and 3"of the hygromycin resistance gene that allows more specific construction of control sequences at the ends of the gene. The third phase tests 4 different arrays of control sequences for their ability to stably transfect the C3 cell line. The arrangement is chosen with minor control sequences that can stably transfect C3 cells as a preferred proviral vector. The retroviral vector pSW272 contains the virus of reticuloendoteliosis (REV), large terminal repeats (LTR), and also selectable marker of thymidine kinase (TK) driven by its own promoter. The LTRs lie at the ends of the provirus, and can function as promoters. By itself, pSW272 is a stable architecture, stable in this case with reference to the ability to generate full-length retroviruses without internal deletions of its genome. The selectable marker is useful for titrating retroviruses in TK cells, but not useful for transfecting the helper cells necessary to generate the retrovirus. Additionally, problems can arise when the expression vector is inserted into pSW272, and the total construct is then transfected into the C3 cell line. The structure of the construction then includes two internal prompts. The 5"or left promoter (in this case the ovalbumin promoter) may be unstable in this environment, significant retroviruses produced from cells transfected with such a construct undergo frequent eliminations in this region (Emerman and Temin, J. Virology 50 (l) : 42-49 (1984)). The same study provides evidence that a structural gene alone in that location is stable and can be expressed by the LTR, eliminating the need for a promoter.

Since the gene can be virtually any structural gene, this can be the selectable marker. The packaging cell line C3 contains endogenous Tk activity and consequently must be cotransfected with a plasmid that confers hygromycin B resistance. The gene encoding hygromycin B phosphotransferase is cloned into the retroviral vector to generate an improved architecture. Then the expression vector is inserted into the only Xba I site, resulting in a stable architecture, elimination of the need for cotransfection and still allows the titration of the virus with CEF cells. Construction of the retroviral vector Construction of pRE ?. A schematic illustrating the construction of the retroviral vector in Figure 3 is indicated. The promoter and structural gene of thymidine kinase are carried in a 2 kb Xba I -Xma I fragment, both of which are unique in pSW272. PSW272 is digested with Xba I and Xma I, and the largest fragment (approximately 7 kb) is recovered from a low melting gel. This fragment is ligated to a synthetic oligonucleotide containing the restriction sites 5. The resulting construct is confirmed, pRE ?, by digestion with Co. I, since Co. I recognizes a site in the synthetic linker, and a second site outside of the DNA retroviral Modification of the hygromycin B phosphotransferase gene Modification to pREP4. The hygromycin B phosphotransferase gene is contained in the plasmid pREP-4, purchased from Invitrogen. However, there are problems with the hygromycin B phosphotransferase gene at both 5 'and 3 A ends At the 5 A end there is an unfavorable sequence surrounding the start codon, specifically a second start codon from external structure upstream 4pb. The 3"end contains both the polyadenylation signal, which may interfere with the retroviral titer, and the LTR of Raus Sarcoma Virus (RSV), which must be deleted.The 3 'end also lacks convenient restriction sites necessary to generate the desired constructs, and with the remodeling, these sites are included.The plasmids are named after the nature of their control signals.For example, the construct containing both a promoter and a polyadenylation signal is designated p ++. plasmid containing a promoter, but one a polyadenylation signal is called p + Construction of p ++ The 3"end of the hygromycin B phosphotransferase gene is modified using synthetic double chain oligos. Figure 12 shows the modification of the 3"end of the Hyg gene. A single Sea I site located 60 bp from the stop codon within the gene (See Figure 12) A synthetic oligonucleotide containing a Sea I site is cloned. C stop codons and stop codon of the hygromycin marker gene, a Hind III site and flanking ends Nsi I and Sal I at the Nsi I and Sal I sites in pREP 4. The appropriate recombinants are verified by double digestion with HindIII - Nru I and no p ++ Construction of p +. P ++ is partially digested with Sea I and the 6.3 kb fragment recovered from a low fusion gel and self-ligated, resulting in a hygromycin construct lacking the 3"control elements except arrest codons (see Figure 12). The appropriate recombinants are verified by double digestion with Sea I - Co. I, and named p + -. Construction of p- + and p--. The N-termination codons are modified in a similar way. Afl III is unique in p ++, and rests just 5"for the start codon of the hygromycin B phosphotransferase gene.A Aat II site rests 25 bases in the hygromycin gene, making double digestion Afl III - Aat II suitable for promoter removal Aat II is not unique to p ++, and thus a strategy of two enzymes is used: p ++ is digested with Cia I and Aat II and in a separate reaction Cia I and Afl III. The digestion products are run on a gel low fusion, and the 5.5 kb Cia I Aat II product is recovered, and the 2.4 kb Cia I Afl III product is recovered.These two DNAs are ligated with a synthetic oligonucleotide (see Figure 13) resulting in p- + Appropriate recombinants are verified by double digestion with Bel AIw NI and sequence analysis The plasmid p + - is treated in the same way, resulting in the p- plasmid.The manipulation is made to the N terminal portion of the gene independently of p ++ , and p + - The four constructions result The testers contain all permutations for control signals such as: 1) with promoter, with poly A signal - contained in a Nru I - Hind III fragment of p ++; 2) with promoter, without poly A signal - contained in a Nru I - Hind III fragment of p + -; 3) without promoter, with poly AD signal - contained in a Bel I - Hind III fragment of p--. Fragments with promoters (1 and 2 above) are cloned into pRE? in the sites Sma I (Xma I) and Hind III in the multiple cloning site. Since secondary recircularization for incomplete digestion is always a concern, is the pRE plasmid digested? in three places: Sma I, Hind III and Acc III. The recovery of the two fragments of the appropriate size from low melting gels assure digestion at both sites within MCS, and when they bind to the Nru I-Hind III fragments in a ligation of molecule 3, it results in totally the desired plasmid. These plasmids are called p ++ R and p + -R. A similar procedure is used to clone the hygromycin B phosphotransferase gene without a promoter in REV. The hygromycin constructs (p- + and p-) are digested with Bel I and Hind III, and cloned in a 3-molecule ligation to gel-purify fragments of Bel I, Hind III and Acc III from pRE ?, and they call p- + R and p-R. The insertion of the expression vector in these retroviral vectors is as follows. Each retroviral vector contains a unique Xba I site. The appropriate plasmid is opened with Xba I, dephosphorylated, and gel purified. pBCWM contains the expression vector in a chloramphenicol-resistant plasmid as a 5 kb Xba I fragment. The pBCMW is digested with the Xba I fragment and the 5 kb fragment of a low fusion gel and ligated to the appropriate retroviral vector. The appropriate recombinants are verified by digestion with Xba I, and orientation is checked by digestion with Eco RI. These plasmids are named as by their retroviral vectors, with the addition of E and the number of the clone. For example: p ++ REl, with promoter, with polyadenylation, in REV and with expression vector in orientation 1. SPECIFIC EXAMPLE 3 - TRANSGENESIS Method 1 Each of the two orientation constructs for a given retroviral vector, are transfected into the cell line C3, and stable clones selected. DNA is isolated from the clones and analyzed by intact proviral DNA composed of Southern biot. Proper clones are propagated and tested by retrovirUs in CEF cells selected for hygromycin resistance. The clones that produce high titers are used to generate retroviruses, which is also concentrated by filtration and centrifugation. When a clone is found to produce high titers of intact viral DNA, the eggs are injected as described in U.S. Patent No. 5,162,215 and Bosselman, R.A. et al., Science 243: 533-535 (1989), incorporated herein by reference. SPF O baseline eggs are obtained from SPAFAS (Preston, CT), and kept at 20 ° C on one side for at least 5 hours. The upper part of the egg is prepared with 70% ethanol, and dried in air. The shell is then opened with a drilling motor-tool with a steel protrusion. 15-25 microliters of a solution containing retrovirus below the blastoderm is microinjected. The eggs are sealed and incubated in a Humidaire incubator until hatched. Ten days after hatching, the blood is collected from the chickens, and tested for the presence of viral DNA in their genomes by Southern biot and PCR. All chickens grow to maturity, at which time the eggs are tested and chicken semen is tested for viral DNA by Southern biot. The positive semen roosters are used to breed G2 chickens which are true heterozygous transgenic chickens. Method 2 Attach the 5 kb insert (the expression vector) from pBCWM to pRE? or p + -, cut with Xba I, dephosphorylated and purified in a low melting gel. The clones are examined for the insert by digestion with Xba I, and orientation is checked by digestion with Eco RI. C3 cells are seeded at 2-3 x 105 cells per well in a 5-well plate (35 mm well diameter) and grown overnight in DMEM with concentrated glucose supplemented with L-glutamine, 10 mM HEPES, 7% calf serum, 400 μg / ml G-418, 100 μg / ml gentamicin, 5 μg / ml fungizone (amphotericin B), 100 units / ml penicillin, G, 100 μg / ml of streptomycin sulfate, at 37 ° C in 10% C02. The cells are transfected using lipofectamine (Gibco Life Technologies) in a proportion of 1.5 μg of DNA per 8 ul of lipofectamine, according to the manufacturer's specifications. After 5 hours the transfection medium is aspirated and replaced with 0.5 ml of DMEM with 7% calf serum, and HEPES. The medium is removed after 48 hours of incubation and used for microinjection or concentrated by ultrafiltration 20 times with a 50 kd cut filter and used for microinjection. Freshly fertilized SPF white paw eggs are placed recently from SPAFAS and are kept on their side for at least 5 hours. A piece of pentagonal shell shape of approximately 0.5 cm2 intact from the uppermost portion of the egg is removed using a fixed Dremmen motor tool with a steel blade (part 113). The membrane of the shell is removed with an 18 gauge needle. The micropipettes are pulled on a Sutter squeegee, cut with a blade, and checked for bore diameter and angle under a microscope. 15 to 20 μl of medium is injected into the subgerminal space using a Narishige micromanipulator (model MN-15) and microinjector (model IM-6). The hole is patched using donor membranes harvested from eggs in the same batch kept briefly in SAF with penicillin G and streptomycin sulfate used in the concentrations stated above. The fragment of the shell is replaced in the upper part of the donor's memebran, and air-dried for 10 minutes. Duco cement is used to seal the edges and air dry for at least 30 minutes. The eggs are then fixed in a Humidaire incubator (model 21) and hatched according to the manufacturer's specifications. Those skilled in the art can now appreciate from the foregoing description that broad teachings of the present invention can be implemented in a variety of ways. Therefore, while this invention has been described in connection with particular examples thereof, the true scope of the invention should not be limited as other modifications will become apparent to an expert practitioner after studying the drawings, specification and claims annexed All patents and other publications cited herein are expressly incorporated by reference.

SEQUENCE LISTING (1) GENERAL INFORMATION (i) APPLICANT: MACARTHUR, William C. (ii) TITLE OF THE INVENTION: Vectors and methods for specific synthesis to protein tissue in eggs of transgenic hens (iii) NUMBER OF SEQUENCES: 31 ( iv) CORRESPONDENCE ADDRESS: (A) RECIPIENT: Harness, Dickey & Pierce, P.L.C. (B) STREET: P.O. Box 828 (C) CITY: Bloomfield Hills (D) STATE: Michigan (E) COUNTRY: E.U.A. (F) POSTAL CODE: 48303 (v) COMPUTER LEADABLE FORM: (A) MEDIUM TYPE: Flexible disk (B) COMPUTER: IBM PC Compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Reread # 1.0, Version # 1.25 (vi) CURRENT REQUEST DATA: (A) APPLICATION NUMBER: PCT / US97 / 09889 (B) SUBMISSION DATE: June 6, 1997 (06.06.97) (C) CLASSIFICATION: (viii) ) INFORMATION OF THE EMPLOYEE / AGENT: (A) NAME: SMITH Esq., DeAnn F.

(B) REGISTRATION NUMBER: 36,683 (C) REFERENCE NUMBER / DOCUMENT: 6550-000008PPB (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (248) 641-1600 (B) TELEFAX: (248) 641-0270 ( C) TELEX: 287637 HARNES UR (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1753 base pairs (B) TYPE: nucleic acid (C) CHAIN: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: fragment (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TCTAGACCAT GGAGCGGAGA ATGGGCGGAA CTGGGCGGAG pAGGGGCGG GATGGGCGG? 60 GTT? GGGGCG GGACTATGGT TGCTGACTAA TTGAGATGCA TGCTTTGCAT ACTTCTGCCT 120 GCTGGGGAGC CTGGGGACTT TCCACACCTG GTTGCTGACT AATTGAGATG CATGCTITGC 180 ATACpCTGC CTGCTGGGGA GCCTGGGGAC TTTCCACACC CTAACTGACA CACATTCCAC 240 AGCAGATCCC CCGGAATTCG GTCAAGCTGA CCTACTAGTG GTCATCATGC ATTTCATAGG 300 TAGAGATAAC ATTTACTGGG AAGCACATCT ATCATCACAA AAAGCAGGCA AGATTTTC? G 360 ACTTTCTTAG TGGCTGAAAT AGAAGCAAAA GACGTGATTA AAAACAAAAT GAAACAAAAA 420 AAATCAGpG ATACCTGTGG TGTAGACATC CAGCAAAAAA ATApATpG CACTACCATC 43J pGTCpAAG TCCTCAGACT TGGCAAGGAG AATGTAGAp TCCACAGTAT ATATG7TTTC 5 ACAAAAGGAA GGAGAGAAAC AAAAGAAAAT GGCACTGACT A? ACpCAGC TAGTGGTATA 600 GGAAAGTAAT TCTGCTTAAC AGAGATTGCA GTGATCTCTA TGTATGTCCT GAAGAApAT 660 GpGTACTTT pTCCCCCAT TpTAAATCA AACAGTGCp TACAGAGGTC AGAATGGTp 720 CpTACTGp TGTCAApCT ApApTCAA TACAGAACAA TAGCTTCTAT AACTGAAATA 780 TApTGCTAT TGTATApAT GApGTCCCT CGAACCATGA ACACTCCTCC AGCTGAATTT 840 CACAATTCCT CTGTCATCTG CCAGGCCAp AAGpATTCA TGGAAGATCT pGAGGAACA 90) CTGCAAGTTC ATATCATAAA CACApTGAA ApGAGTAp GTTTTGCAp GTATGGAGCT 960 ATGTTpGCT GTATCCTCAG AAAAAAAGp TGpATAAAG CApCACACC CATAAAAAGA 1020 TAGATpAAA TATTCCAACT ATAGGAAAGA AAGTGTGTCT GCTCpCACT CTAGTCTCAG 1GS0 TTGGCTCCTT CACATGCACG CpCTpAp TCTCCTA1TT TGTCAAGAAA ATAATAGGTC 1140 AAG ~ CTTGp CTCATpATG TCCTGTCTAG CGTGGCTCAG ATGCACApG TACATACAAG 1200 AAGGATCAAA TGAAACAGAC TTCTGGTCTG pACTACA? C CATAGTAATA AGCACACTAA 1360 CTAATAApG CTAApATGT pTCCATCTC CAAGGpCCC ACATTITICT GTpTCTTAA 1333 AGATCCCATT ATCTGGpGT AACTGAAGCT CAATGGAACA TGAGCAATAT pCCCAGTCT 1380 TCTCTCCCAT CCAACAGTCC TGATGGApA GCAGAACAGG CAGAAAACAC ApGTTACCC 1440 AGAApAAAA ACTAATApT GCTCTCCATT CAATCCAAAA TGGACCTAp GAAACTAAAA 1500 TCTAACCCAA TCCCATTAAA TGATTTCTAT GGTGTCAAAG GTCAAACpC TGAAGGGAAC 1560 CTGTGGGTGG GTCACAApC AGACTATATA TTCCCCAGGG CTCAGCCAGT GTCTGTACCT 1620 ACA3CTAGAA AGCTGTApG CCpTAGCAC TCAAGCTCAA AAGA CAACTC AGAGpCACC 1680 TGpACATAC AGCTATGAGG TCpTGCTAA TCpGGTGCT pGCpCCTG CCCCTGGCTG 1740 CTCTGGGGAA TAT 1753 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) CHAIN: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Arg Ser Leu Leu lie Val Leu Cis Fen Leu Pro Leu 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) CHAIN: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: fragment (ii) HYPOTHETICAL: NO (iii) ANTISENTIDO: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GTACATACAG CTATGAGGTC TTTGCTAATC TTGGTGCTTT GCTTCCTGCC CCTG 54 (2) INFORMATION FOR SEQ ID NO: 4 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) CHAIN: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: fragment (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: CAGGGGCAGG AAGCAAAGCA CCAAGATTAG CAAAGACCTC ATAGCTGTAT 50 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF SEQUENCE: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 5: Ala Ala Leu Gli Asn lie l 5 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) CHAIN: one (D) TOPOLOGY: linear (ii) TYPE MOLECULE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GCTGCTCTGG GGAATAT 17 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) CHAIN: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: fragment (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) ) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CGATATTCCC CAGAGCAGC 19 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) CHAIN: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: fragment (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 8: CGTTATCTTT CGAAGGTGTC GTTTTACAAC GTCGTGAC 38 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) CHAIN: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: fragment (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 9: ACCACCGCGA CCTACCATTC GGCG 24 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) CHAIN: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: fragment (iü) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GATCTACCGC GGACGGTACC TACAGCTAGA AAGCTGTATT GCCTTTAGCA 50 (2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS SEQUENCE: (A) LENGTH: 46 base pairs (B) TYPE: nucleic acid (C) CHAIN: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: fragment (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11 TGCTAAAGGC AATACAGCTT TCTAGCTGTA GGTACCGTCC GCGGTA 46 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) CHAIN: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: FRAGMENT (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: CTCAAGCTCA AAAGACAACT CAGAGTTCAC CT 32 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) CHAIN: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: fragment (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 13: GTACAGGTGA ACTCTGAGTT GTCTTTTGAG CTTGAG 36 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) CHAIN: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: fragment (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GTACATCTAG ACCATGGTCC GGACAGCGAA ATGGAGAATT CGGTCAAGCT TCACTGACCT 60 GACTAGTGGT CATCATGCA 79 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) CHAIN: one (D) TOPOLOGY: linear (ii) TYPE MOLECULE: fragment (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: TGATGACCAC TAGTCAGGTC AGTGAAGCTT GACCGAATTC TCCATTTCGC TGTCCGGACC 60 ATGGTCTAGA T 71 (2)) INFORMATION FOR SEQ ID NO: 16 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) CHAIN: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: fragment (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: GTACCTCTAG ACCATGGTCC GGACAGCTCA ATGGAGAATT CGGTCAAGCT TCACTGGCCT 60 GACTAGTGGT CATCATGCA 79 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) CHAIN: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: fragment (iii) HYPOTHETICAL: NO ( iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: TGATGACCAC TAGTCAGGCC AGTGAAGCp GACCGAATTC TCCApGAGC TGTCCGGACC 60 ATGGTCTAGA G 71 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base pairs (B) TYPE: nucleic acid (C) CHAIN: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: FRAGMENT (iü) HYPOTHETICAL: NO (iv) ANTISENTIDO: NO (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 18: GTACCTCTAG ACCATGGTCC GGACAGCTCA ATGGAGAATT CGGTCA 46 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) CHAIN: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: fragment (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: AGCTTGACCG AATTCACCAT TGAGCTGTCC GGACCATGGT CTAGAGGTAC 50 (2) INFORMATION FOR SEQ ID NO: 20: (i) CHARACTERISTICS SEQUENCE: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: fragment (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 20 CTAGTGGTCA TCATGCA 17 (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 base pairs (B) TYPE: nucleic acid (C) CHAIN: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: fragment (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 21: AGCATGATGA CCA 13 (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 base pairs (B) TYPE: nucleic acid (C) CHAIN: one (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: fragment (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 22 AGCTGACCTA 10 (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 base pairs (B) TYPE: nucleic acid (C) CHAIN: one (D) TOPOLOGY: linear (ii) TYPE MOLECULE: fragment (v) HYPOTHETICAL: NO (vi) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: CTAGTAGGTC 10 (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: ( A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) CHAIN: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: fragment (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: GAATTCGGTC AAGCTGACCT ACTAGTGGTC ATCATGCA 38 (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) CHAIN: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: fragment (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: TGCATGATGA CCACTAGTAG GTCAGCTTGA CCGAATTC 38 (2) INFORMATION FOR SEQ ID NO: 26: (i) CHARACTERISTICS OF SEQUENCE: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) CHAIN: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: fragment (iii) HYPOTHETICAL: NO (iv) ANTISENTIAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: CTAGTCCCGG GTGATCATCG ATTGAAGCTT TCTAGAA 37 (2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) CHAIN: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: FRAGMENT (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 27: CCGGTTCTAG AAAGCTTCAA TCGATGATCA CCCGGGA 37 (2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) CHAIN: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: fragment (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: CGCGATGATC AGTCACCATG AAAAAGCCTG AACTCACCGC GACGT 45 (2) INFORMATION FOR SEQ ID NO: 29: (i) CHARACTERISTICS OF SEQUENCE: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) CHAIN: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: fragment (üi) HYPOTHETICAL: NO (iv) ANTISENTIDO: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: CGCGGTGAGT TCAGGCTTTT TCATGGTGAC TGATCAT 37 (2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) CHAIN: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: fragment (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: TCGACAGTAC TCGCCGATAG TGGAAACCGA AGACCATCTA CACGACCGAA GTCAAAGGAA 60 TAGTAGAAGC TTATGCA 77 (2) INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 69 base pairs (B) TYPE: nucleic acid (C) CHAIN: one (D) TOPOLOGY: linear (ii) TYPE MOLECULE: fragment (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: TAAGCpCTA CTApCCpT GACpCGGTC GTGTAGATGG TCpCGGpT CCACTATCGG 60 CGAGTACTG &

Claims (45)

1. CLAIMS 1. A retroviral vector defective in replication, characterized in that it comprises: a) a transgene; and b) control elements operably linked to the transgene and capable of directing the synthesis of the transgene product in a bird white egg, wherein the control elements comprise a promoter, a 5"untranslated region and a signal sequence. The vector according to claim 1, characterized in that the retroviral vector defective in replication is derived from REV-A 3. The vector according to claim 1, characterized in that the promoter of the group consisting of ovalbumin promoters is chosen. , lysozyme, conalbumin and ovomucoids, and combinations thereof 4. The vector according to claim 1, characterized in that the bird is a chicken 5. The vector according to claim 1, characterized in that the control elements also they comprise an amplifier 6. The vector according to claim 3, characterized in that the promoter comprises an ovalbumin promoter. 7. The vector according to claim 5, characterized in that the amplifier comprises a spheroid hormone response element. 8. The vector according to claim 5, characterized in that the amplifier is a viral amplifier. 9. The vector according to claim 8, characterized in that the amplifier is a portion of SV40. 10. A retroviral vector defective in replication characterized in that it comprises: c) a transgene; and d) control elements operably linked to the transgene and capable of directing the synthesis of the transgene product in the bird egg yolk, wherein the control elements comprise a promoter, a non-translated region, a signal sequence and a sequence of catchment. 11. The vector according to claim 10, characterized in that the retroviral vector defective in replication is derived from REV-A. 12. The vector in accordance with the claim 10, characterized in that the promoter of the group consisting of vitellogenin and apolipoprotein A promoters and combinations thereof is chosen. 13. The vector according to claim 10, characterized in that the bird is a chicken. 14. The vector according to claim 10, characterized in that the control elements also comprise an amplifier. 15. The vector according to claim 14, characterized in that the amplifier comprises a spheroid hormone response element. 16. The vector according to claim 14, characterized in that the amplifier is a viral amplifier. 17. The vector in accordance with the claim 16, characterized in that the amplifier is a portion of SV40. 18. A method for transferring a transgene to an embryonic chicken cell, characterized in that it comprises the step of introducing a retroviral vector defective in the replication in the cell, wherein the vector comprises the transgene and control elements operatively linked to it and capable of of directing the synthesis of the transgene product in white egg, wherein the control elements comprise a promoter, a 5"untranslated region, and a signal sequence 19. The method according to claim 18, characterized in that the vector replication defective retroviral is derived from REV-A 20. The method according to claim 18, characterized in that the control elements further comprise an amplifier 21. The method according to claim 18, characterized in that the promoter of the group consisting of promoters of ovalbumin, lysozyme, conalbumin and ovomucoids, and combinations thereof. 22. The method according to claim 20, characterized in that the amplifier comprises a spheroid hormone response element. 23. The method according to the claim, characterized in that the amplifier is a viral amplifier. 24. The method according to claim 23, characterized in that the amplifier is a portion of SV40. 25. A method for transferring a transgene to an embryonic chicken cell, characterized in that it comprises the step of introducing a retroviral vector defective in replication in the cell, wherein the vector comprises the transgene and control elements operatively linked to it and capable of of directing the synthesis of the transgene product in egg yolk, wherein the control elements comprise a promoter, an "untranslated" S region, and a signal sequence 26. The method according to claim 25, characterized in that the Replicating defective retroviral vector is derived from REV-A 27. The method according to claim 25, characterized in that the control elements further comprise an amplifier 28. The method according to claim 25, characterized in that the promoter of the group consisting of vitellogenin and apolipoprotein A promoters, and combinations thereof, is chosen. 29. The method according to claim 27, characterized in that the amplifier comprises a spheroid hormone response element. 30. The method according to claim 27, characterized in that the amplifier is a viral amplifier. 31. The method of compliance with the claim 30, characterized in that the amplifier is a portion of SV40. 32. A method for producing a transgenic chicken characterized in that it comprises the steps of: a) Making an opening in a chicken egg which stops at stage X and which contains an embryo, to thereby expose the blastoderm. b) Microinject through the opening in an area in close proximity to the blastoderm, a solution containing a retroviral vector defective in the replication comprising a transgene and control elements operably linked thereto and capable of directing the synthesis of the transgene product in white egg, where the control elements comprise a promoter, a 5"untranslated region and a signal sequence, c) Seal the opening after the microinjection, ed) Incubate the egg until the chicken hatches from the egg. The method according to claim 32, characterized in that the retroviral vector defective in replication is derived from REV-A 34. The method according to claim 32, characterized in that the control elements further comprise an amplifier. The method in accordance with the claim Characterized in that the promoter of the group consisting of ovalbumin, lysozyme, conalbumin and ovomucoid promoters, and combinations thereof is chosen. 36. The method according to claim 34, characterized in that the amplifier comprises a steroid hormone response element. 37. The method according to claim 34, characterized in that the amplifier is a viral amplifier. 38. The method according to claim 37, characterized in that the amplifier is a portion of SV40. 39. A method for producing a transgenic chicken characterized in that it comprises the steps of: a) Making an opening in a chicken egg which is no more than seven days old and which contains an embryo, to thereby expose the blastoderm. b) Microinject through the opening in an area in close proximity to the blastoderm, a solution containing a retroviral vector defective in the replication comprising a transgene and control elements operably linked thereto and capable of directing the synthesis of the transgene product in white egg, wherein the control elements comprise a promoter, a 5"untranslated region and a signal sequence, c) Seal the opening after the microinjection, ed) Incubate the egg until the chicken hatches from the egg. The method according to claim 39, characterized in that the retroviral vector defective in replication is derived from REV-A 41. The method according to claim 39, characterized in that the control elements further comprise an amplifier. The method according to claim 39, characterized in that the promoter of the group consisting of vitellogenin promoters is chosen. a and apolipoprotein A, and combinations thereof. 43. The method according to claim 41, characterized in that the amplifier comprises a spheroid hormone response element. 44. The method according to claim 41, characterized in that the amplifier is a viral amplifier. 45. The method according to the claim 44, characterized in that the amplifier is a portion of SV40.