KR100921226B1 - Retrovirus expression vector containing ??-??? human granulocyte-colony stimulating factor gene and Transgenic poultry thereby - Google Patents

Abstract

The present invention relates to retrovirus vectors for transduction and expression of genes encoding human Granulocyte-colony Stimulating Factor of human blood, poultry transformed by them, and methods for producing the same.

According to the retroviral vector of the present invention, by efficiently transferring human hG-CSF gene, cells and poultry of various animals can be efficiently transformed to express the hG-CSF gene. Can be used as a bioreactor to economically mass produce hG-CSF.

Poultry, transformation, replication-defective retrovirus, human Granulocyte-Colony Stimulating Factor (hG-CSF)

Description

Retroviral expression vector containing human granulocyte-colony stimulating factor gene and transgenic poultry according to which granule colony stimulating factor gene of human blood is incorporated

The present invention relates to retrovirus vectors for transduction and expression of genes encoding human Granulocyte-colony Stimulating Factor of human blood, poultry transformed by them, and methods for producing the same.

Human granulocyte colony stimulating factor (hG-CSF) is a 19.6 kd glycoprotein that regulates the proliferation and differentiation of hematopoietic progenitor cells (Bober et al., 1995, Immunopharmacology 29: 111-119), solid cancer and blood tumors It is used to treat neutropenia in patients with chemotherapy, onset of aplastic anemia, myelodysplastic syndrome, and hematopoietic stem cell transplantation (Kaushansky, 2006, N. Engl J. Med. 354: 2034-2045). . Most of the currently used hG-CSF is produced in recombinant protein form mainly in Escherichia coli. However, proteins produced in E. coli are limited in many ways because they have many problems in posttranslational modifucation, such as glycosylation. The most ideal way to solve this problem is to first harvest the glycoprotein (glycoprotein) directly from the hG-CSF gene is introduced and expressing the animal, that is, the transgenic livestock.

Until recently, most of the research on the production of transgenic livestock was carried out mainly on mammals such as cattle, pigs, sheep, rabbits and goats. Despite their large investments in time and money, there are few reports on the successful economic production of transgenic livestock. This is largely due to the long generation intervals of mammals, the complex physiological morphological features, and the astronomical costs of research.

On the other hand, poultry has a shorter maturation period and generation intervals than mammals, and has a very high reproductive capacity compared to mammals, making it easy to establish transformed poultry and experimenting with large numbers of individuals with relatively little effort and cost. (Ivarie, 2003, Trends Biotechnol. 21: 14-19).

However, unlike in mammals, poultry development is due to the general polyspermy phenomenon (Perry, 1987, J. Anat. 150: 99-109), which is different from mammals due to embryonic development from fertilization to post-production. There are already about 60,000 pluoripotent cells in the embryos of freshly laid eggs (stage X, Eyal-Giladi et al., 1976, Dev. Biol. 49: 321-337) (Petitte and Mozdziak, 2002, in: Transgenic Animal Technology (edited by CA Pinkert, Chapter 11, Academic Press, San Diego) .Because of these morphological and developmental characteristics, introducing foreign genes in poultry in the same way as in mammals It is also very difficult to obtain early fertilized eggs from poultry eggs, as well as the eggs of the eggs that are spawned with thick eggshells. Application of the gene transfer technology used in mammals is impossible.

Target cells used for the introduction of foreign genes in the production of transgenic poultry include egg x stage blastocyst cells (Rosenblum and Chen, 1995, Transgenic Res. 4: 192-198), primordial germ cells. (Li et al., 1995, Transgenic Res. 4: 26-29), freshly fertilized eggs (Sherman et al., 1998, Nat. Biotech. 16: 1050-1053), embryonic stem cells; Pain et al., 1999, Cells Tissues Organs 165: 212-219) and sperm (Nakanishi and Iritani, 1993, Mol. Reprod. Dev. 36: 258-261), and DNA target cells for the transfer of foreign genes. Biological methods using either physical or chemical methods (Muramatsu et al., 1997, Biochem. Biophys. Res. Commun. 230: 376-380) or directly infecting the virus (Thoraval et al. , 1995, Transgenic Res. 4: 369-377). Therefore, there are many methods for producing transgenic chickens, depending on the target cells and gene transfer methods to transfer foreign genes. Among them, the retroviral vector system is used for X stage blastocyst cells. Is being used. The biggest advantage of this method is that it is technically the easiest because it uses the inherent infectivity of retroviruses in gene transfer. Using this method, Salter et al. (1987, Virology, 157: 236-240) were the first to inject a replication-competent retrovirus vector directly into chicken embryos to confirm the transfer of foreign genes in germ cells. Transgenic chickens were produced, and then Bosselman et al. (1989, Science, 243: 533-535) produced transgenic chickens using a replication-defective retrovirus vector. Recently, McGrew et al. (2004, EMBO reports, 5: 728-733), Lillico et al. (2007, Proc. Natl. Acad. Sci. USA, 104: 1771-1776), Harvey et al. (2002, Nat. Biotech. , 19: 396-399), Mozdziak et al. (2003, Dev. Dyn. 226: 439-445) and Rapp et al. (2003, Transgenic Res. 12: 569-575). However, these studies have the following disadvantages. In other words, these studies used lentivirus vector systems such as EIAV (equine infectious anemia virus) and HIV-1 (human immunodeficiency virus type 1), or ALV (avian leukosis virus), REV (reticuloendotheliosis virus) or SNV (spleen necrosis virus) and The same avian retrovirus system was used. The lentivirus vector represented by HIV has inherent biological safety issues, and the use of the avian retrovirus vector is inherent in transfected viral vectors and chicken cells. Endogenous retroviruses may be subjected to homologous recombination, which may present a biohazard problem in which viruses can be produced.

In order to solve this problem, the present inventors have disclosed a method for transforming poultry using a replication-deficient retrovirus expression vector derived from murine leukemia virus (MLV) in Korean Patent No. 696571. Since MLV is not an algae-derived retrovirus, there are few biological risk factors as described above, and it is easy to obtain a high concentration of retroviral vector stock, which enables efficient gene transfer.

As compared to other mammals, the transformation of poultry has many limitations, and thus, the production of active substances by the transformation of poultry has not been studied yet.

The present invention is to solve the problems of the prior art as described above, an object of the present invention is to provide a retroviral vector that is easy to transfer and expression of human hG-CSF gene and low biological risk.

In another aspect, the present invention is to provide a method for transforming poultry having a high reproduction capacity, short maturity and generation intervals compared to mammals using the retroviral vector, and to provide a poultry transformed thereby.

The present invention for achieving the above object is characterized in that the expression retrovirus (retrovirus) vector containing the recombinant hG-GSF gene of SEQ ID NO: 1.

The advantages of the retrovirus vector system used in the present invention are genetically stable compared to other foreign gene introduction methods (Temin, Genome 31: 17-22, 1989), and do not repeat when the foreign gene is inserted into the genome of the target cell. Only a single gene can be selectively introduced into the chromosome region, and it is possible to infect various kinds of cells with various kinds of foreign genes with high efficiency (Rohdewohld et al., J. Virol. 61: 336-). 343, 1987).

The hG-CSF gene of SEQ ID NO: 1 to be expressed in the present invention is located at 3 'of the CMV (cytomegalo virus) promoter introduced into the retroviral vector so that the expression of the hG-CSF gene occurs under the control of the CMV promoter. To maximize the expression of the hG-CSF gene, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) DNA fragment was introduced at the 3 'position of the hG-CSF cDNA (FIG. 1). .

The retroviral vector may be avian leukosis virus (ALV), reticuloendotheliosis virus (REV), spleen necrosis virus (SNV), or murine leukemia virus (MLV) system, and can be freely replicated in infected cells. It is desirable to use a replication-deficient retroviral system in which the gag, pol and env genes involved in particle formation and replication have been removed to avoid the problem of reproduction and infecting other cells. In addition, poultry transformation involves endogenous retroviruses that are inherent in transfected viral vectors and in poultry cells, even if the same avian retrovirus system uses replication-deficient virus vectors that do not replicate themselves. It is more preferred that the MVL-derived replication-deficient retroviruses be present because biological problems may arise where homologous recombination may produce a virus capable of replication.

In an embodiment of the present invention, a WPRE isolated from pGEM-Teasy-WPRE is introduced into pUC18-GCSF to clone pUC18-GCSFW and to process Spe I, Mungbean nuclease, and Hind III enzyme in sequence to connect hG-CSF cDNA and WPRE. DNA fragment of number 2 was isolated. Site-specific DNA cleavage and binding in this process was performed by treatment of the restriction enzymes described above under conditions generally known in the art. The isolated hGCSF-WPRE Is a MLV-derived replication-deficient virus vector sequentially processed by Cla I, Mungbean nuclease, and Hind III By ligation to pLNCX, pLNC-GCSFW plasmid was finally constructed. The cleavage map of the plasmid pLNC-GCSFW is as shown in FIG. 2. LNC-GCSFW which is a viral vector for expression according to the present invention On September 13, 2007, it was deposited with KCTC (Korean Collection for Type Cultures, KCTC), 52, Eeun-dong, Yuseong-gu, Daejeon, Korea.

Another aspect of the invention relates to an animal cell transformed with said retroviral vector LNC-GCSFW. The animal cells include not only animal cells of poultry, but also animal cells of mammals such as cattle, pigs or mice. More specifically, it is preferable that they are fetal fibroblasts, chicken embryo fibroblasts, HeLa cells as human cells, or NIH3T3 cells as mouse cells as described in the Examples.

Transformation of animal cells with the vectors of the invention is carried out using retroviruses obtained by transferring and expressing MLV-derived replication-deficient retroviral vector LNC-GCSFW to packaging cells. More specifically, LNC-GCSFW, a retroviral vector, is prepared to metastasize and express to PT67 packaging cells (ClonTech, USA) and treated with G418 to select transformed cells. Virus stocks are recovered from selected cells and infected again with GP2-293 cells (ClonTech, USA). Transfecting and expressing pVSV-G (ClonTech, USA) on the infected GP2-293 cells, and then transformed into a fresh medium, the animal cells were infected by using a culture medium obtained by culturing or centrifugation and filtration and concentrated virus stock. Let's do it. Infected cells are treated with G418 to obtain transformed cells.

As a result of measuring the concentration of hG-CSF in the culture medium of the transformed animal cells by ELISA method, it was found that hG-CSF was expressed in the culture medium of all animal cells. In particular, in the culture of transformed chicken cells, the presence of hG-CSF at a concentration of about 3mg / ml confirmed that the animal cells transformed by the present invention can be used as a bioreactor for economic mass production of hG-CSF Could.

Another aspect of the present invention relates to a transgenic poultry characterized in that the retroviral vector LNC-GCSFW can be transformed to express the hG-CSF gene.

As used herein, "poultry" refers to livestock among birds and includes chickens, ducks, turkeys, geese, quails and pigeons.

The ex-ovo culture method (Petitte and Mozdziak, 2002, Transgenic Animal Technology, second ed., Academic Press, San Diego, 2002, pp. 279-306) was used as a method of injecting and hatching viruses into eggs. Chicken embryos include stage I through stage IV, including the clevage stage (from stage I to VI), the formation of the a.pellucida stage (from stage VII) and morphogenetic development) step (from step XI to step XIV). In the present invention, the "embryo" is an early stage of development, in an embryonic or egg-bearing animal, until the end of individual development from the two-cell stage and hatching (hydrolyzing or destroying the fertilized membrane and egg film) to become larvae. It is the time when viral infection occurs for production of transgenic poultry. The step of infecting with viral particles in the present invention is preferably wheezing, which is the last step of the eggplant. The streak is freshly laid and the first signs of endoderm formation appear and clusters of small cells forming a net-like layer in the posterior region of the blastocyst.

A total of 140 eggs were injected with the same recombinant LNC-GCSFW virus as used for the transformation of animal cells to hatch 17 Go transgenic chicks and confirmed that hG-CSF was expressed in the blood of all hatched chicks. Among them, the concentration of hG-CSF in the blood of 13 chicks was confirmed by ELISA method.

The biological activity of hG-CSF produced from transgenic chickens is significantly higher than that produced in Escherichia coli, so that the method of producing hG-CSF from the transformed poultry according to the present invention was produced from conventionally transformed E. coli. It was proved that the effect is excellent.

Also hG-CSF gene was introduced in the transition chicken (Go) transformed by the present invention are oil through the gonads in the next-generation G chicken of _{the first} generation, the new hG-CSF transformed not just a transgenic fowl by this invention It was confirmed that breeding of convert poultry could be established.

Although not described in the examples of the present invention, it can be expected that the eggs produced by the poultry transformed by the present invention also contain hG-CSF. Therefore, eggs of poultry transformed by the present invention will be more conveniently used as a source of unique hG-CSF, unlike mammals.

As described above, according to the retroviral vector of the present invention, the human hG-CSF gene can be efficiently transferred, and cells and poultry of various animals can be efficiently transformed to express the hG-CSF gene. In addition, transformed animal cells or poultry can be used as bioreactors to economically mass produce hG-CSF.

The present invention will be described in detail through the following examples. However, these examples are for illustrative purposes only and the present invention is not limited thereto.

Example 1: Construction of a retroviral vector expressing hG-CSF protein under CMV promoter control ( Plasmid pLNC - GCSFW Build)

According to the method described in Korean Patent No. 696571, the inventors constructed a WPRE sequence (Donello et al., 1998, J Virol. 72: 5085-5092) into the plasmid pGEM-Teasy (purchased from Promega, USA). Plasmid pGEM-Teasy-WPRE was treated with EcoRI enzyme, and the WPRE sequence was isolated and introduced into pUC18-GCSF (purchased from Chonbuk National University cytokine bank) treated with the same EcoR I enzyme to construct and cloning pUC18-GCSFW. The cloned pUC18-GCSFW was treated with Spe I, Mungbean nuclease, and Hind III enzyme in order to isolate DNA fragments linked with hG-CSF cDNA and WPRE sequence. pLNC-GCSFW plasmid (7914 bp) was finally constructed by ligation to pLNCX (Miller and Rosman, 1989, Biotechniques 7: 980-990) treated with nuclease and Hind III (FIG. 1). The cleavage map of the plasmid pLNC-GCSFW is as shown in FIG. 2.

The expression vector, pLNC-GCSFW, was assigned a deposit accession number KCTC 11200BP to KCTC (Korean Collection for Type Cultures, KCTC), an international depository institution, on September 13, 2007.

Example 2 Expression of hG-CSF in Various Animal Cells Transfected by LNC-GCSFW Retrovirus

The retroviral vector pLNC-GCSFW was transferred to PT67 packaging cells (Clontech, USA) and expressed and cultured for two weeks in a selection solution to which G418 (800 μg / ml) was added. Virus stocks harvested from PT67 cells surviving G418 containing screening solutions were infected with GP2-293 (Clontech, USA) cells and screened for 2 weeks with screening solutions added with G418 (800 μg / ml). Of 20 ㎍ the calcium phosphate method for the screening G418- resistant GP2-293-LNC-GCSFW cell pVSV-G (Clontech, USA) and expressed as a transition to 8 hours after incubation at 37 ℃, 5% CO ₂ condition as a new culture medium Changed After 48 hours, the culture solution containing the retrovirus was harvested.

The harvested culture was filtered using a 0.45 μm pore-sized cellulose acetate filter, followed by bovine fetal fibroblasts, chicken fetal fibroblasts, and human cells. HeLa cells, rat NIH3T3 cells, and porcine fetal fibroblasts were infected.

Infected cells were screened for 2 weeks with G418 (800 μg / ml) -added selection solution, and then cultured cultures of selected G418-resistant cells were obtained. Measured together. 100 μl of the recombinant hG-CSF (Komabiotech, Korea), the control and experimental cells of the standard and the experimental group were added to 100 wells of 96 well plates coated with anti-human G-CSF monoclonal antibody (BD Biosciences, USA) the day before. After 16 hours of reaction at C, the cells were washed with water, and then treated with biotinylate rat anti-human G-CSF monoclonal antibody (BD Biosciences, USA). After washing again, streptavidin-HRP conjugate (BD Biosciences, USA) solution was added to each well and reacted. After washing again, TMB (tetramethylbenzidine, Komabiotech, Korea) was added and the substrate was reacted for 20 minutes under dark conditions. I was. After the reaction, the absorbance was measured at a wavelength of 450 nm using a microplate reader, and then the final concentration was determined by comparing the absorbance of standard hG-CSF. As can be seen in Figure 3, it was confirmed that the most produced hC-GSF in the cells of the chicken of the various animal cells. "LNC-GCSFW" following the cell name in the X axis of Figure 3 means that the LNC-GCSFW virus has been transferred to the cell.

Example 3 Production of Transgenic Chickens Expressing the hG-CSF Gene by the Retroviral Vector LNC-GCSFW

First, a highly concentrated virus stock to be injected into eggs was prepared in the following manner. A virus stock of 100 ml of the culture medium of the G418-resistant GP2-293-LNC-GCSFW cells into which the gene of the retroviral vector prepared in Example 2 was introduced was subjected to 90 minutes at 16,700 rpm at 4 ° C. using a vertical rotor (Beckman 70Ti). After centrifugation, the supernatant was completely removed, and 100 ml of 0.1 X Han's Balanced Salt Solution (HBSS) or DMEM (Dulbecco's Modified Eagle's Medium) was added to the precipitate, which was left for 16 hours at 4 ° C and resuspended. As a result, the virus stock concentrated at about 1,000-fold was filtered using a 0.45 μm pore-size cellulose acetate filter and stored at −70 ° C. before use.

The ex-ovo culture method (Petitte and Mozdziak, 2002, Transgenic Animal Technology, second ed., Academic Press, San Diego, 2002, pp. 279-306) was used as a method of injecting and hatching viruses into eggs. In other words, transfer the contents of the eggs to a peptri dish and inject 5 µl of concentrated virus solution containing polybrene (10 ㎍ / ml) into the embryo. The contents of the virus-injected eggs are again transferred to an empty eggshell with a diameter of about 33 mm, sealed with a parafilm, and placed in an incubator at a temperature of 37.7 ℃ and a relative humidity of 60%. Occurs during eggplant for 3 days. On the fourth day of development, the contents of the eggs are transferred to another empty egg shell and then under the condition of 37.7 ° C. and 70% relative humidity until hatching. The egg was not fertilized from the 19th day of development, and from the 20th day of development, the parafilm sealing the eggshell was replaced with a petridish cap to make it easier for the chicks to escape from the eggshell.

As a result, a total of 140 eggs were injected with recombinant LNC-GCSFW virus to incubate 17 Go transgenic chicks, and it was confirmed that hG-CSF was expressed in the blood of all hatched chicks, 13 of which were chicks. The concentration of hG-CSF in blood was confirmed by ELISA method (Fig. 4). The unique number and sex of Go chickens subjected to ELISA under the X axis of FIG. 4 are shown.

Example 4 Comparison of Biological Activity of hG-CSF Produced from Chickens

The biological activity of hG-CSF produced from transgenic chickens was compared by measuring the extent of proliferation of MNFS-60 cells.

First, 2.5 × 10 ⁴ MNFS-60 cells were added to each well of a 96 well plate with 50 μl RPMI 1640 and 50 μl chicken serum samples (or Escherichia coli-produced concentrations of hG-CSF added 5% Fetal Calf Serum). Incubated for 18 hours in a mixed medium (purchased by Strathmann Biotech, Germany), and then tritiated thymiodine (0.5 μCi / well) was added to each well and incubated for 4 hours, and then the collected cells were transferred to a scitillation counter. The amount of radiation was measured, and as a result, hG-CSF (indicated by "-○-○-○-") produced in transgenic chickens was expressed as hG-CSF ("-● ― ● ― ● ―") produced by E. coli. It was confirmed that the biological activity is significantly higher than) (Fig. 5).

Example  5: hG - CSF  Germline transmission of genes

Nine of the 17 Go chickens expressing the hG-CSF gene were male. The sperm inserted into the chromosome of the hG-CSF gene in these semen of 9 males was examined by PCR.

As primer pairs specific for the hG-CSF gene, 5'- AGAAGCTGTGTGCCACCTACAAGC-3 'of SEQ ID NO: 3 and 5'-GTACGACACCTCCAGGAAGCTCTG-3' of SEQ ID NO: 4 were used. The total number of PCR reaction cycles was 35, and the reaction mixture was reacted for 30 seconds at 94 ° C, 30 seconds at 54 ° C, and 30 seconds at 72 ° C for each cycle. The mixture for the PCR reaction (50 μl) contains 1 μg genomic DNA, 10 pmol of each primer, 5 μl of 10X PCR buffer (purchased from Promega, USA), 1.5 mM MgCl ₂ , 0.2 mM dNTP each, and 2.5 U. Taq polymerase (purchased from Promega) was prepared by mixing. A total of 398 bp amplified DNA bands were confirmed in 6 samples among 9 male semen samples (FIG. 6). In FIG. 6, "M" indicates molecular size markers, "P" and "N" indicate PCR of plasmid pLNC-GCSFW and semen from non-transgenic chicken, respectively. The numbers above each lane represent the unique numbers of each rooster.

As a control, primer pairs (5'-TGATGCCCCCATGTTTGTGA-3 ') (SEQ ID NO: 5) and 5'-CAAGAAGGGAACACGCAGGG-3' (SEQ ID NO: 6) for the glyceraldehydes phosphate dehydrogenase (GAPDH) gene were used. band appeared.

As a result, it was confirmed that the hG-CSF gene was transferred to 6 gonads among 9 male Go trans chickens.

Example  6: LNC - GCSFW  Transferred by retroviral vectors hG - CSF  Heredity to the next generation of genes

Southern blot was used to determine whether the hG-CSF gene transferred by Go transgenic chickens by the LNC-GCSFW retroviral vector is also inherited by chickens of the next generation, G1. The Southern blot probe is a 541 bp DNA produced by PCR using primer pairs specific for the hG-CSF gene (5'-CATGAAGCTGATGGCCCTGC-3 '(SEQ ID NO: 7) and 5'-GCTCTGCAGATGGGAGGCAA-3' (SEQ ID NO: 8)). fragment was used.

DNA from the blood of three G ₁ chickens produced in the mating of 003 roosters and non-transgenic hens of FIG. 6 was treated with Hind III restriction enzyme followed by Southern blot, and in one hG-CSF gene, Two copies of each cell were confirmed to exist 1 copy of each cell in the remaining two (Fig. 7). In FIG. 7, M is a molecular size marker, P is Southern blot after treatment with palsmid pLNC-GCSFW with Hind III, and N is Southern blot after treatment with blood DNA of a normal chicken, not a transgenic chicken, with Hind III. . 1 to 3 above each lane are Southern blot after Hind III treatment of blood DNA of each G ₁ chicken.

1 is a schematic flowchart showing a manufacturing process of pLNC-GCSFW.

2 is a cleavage map of the plasmid pLNC-GCSFW.

Figure 3 shows the expression of the hG-CSF gene in the cells of bovine (BFF), chicken (CEF), human (HeLa), rat (NIH3T3) and pig (PFF), which gene has been transferred by the retroviral vector LNC-GCSFW. Showing graph.

4 is a graph showing the concentration of hG-CSF in the blood of chickens transformed with the retroviral vector LNC-GCSFW.

5 is a graph showing a comparison of the biological activity of hG-CSF produced in transgenic chickens (-○-○-○-) and E. coli ( E. coli ) (-●-●-●-).

6 is a PCR result picture showing the presence of hG-CSF gene in the semen of the transformed Go rooster.

7 is a Southern blot result photograph showing that the hG-CSF gene transferred to transgenic chickens is inherited by G1 generation.

<110> SUN MOK INSTITUTE EDUCATION FOUNDATION <120> Retrovirus expression vector containing hG-CSF (human          granulocyte-colony stimulating factor) gene and transgenic          poultry thereby <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 615 <212> DNA <213> hG-CSF <400> 1 atggctggac ctgccaccca gagccccatg aagctgatgg ccctgcagct gctgctgtgg 60 cacagtgcac tctggacagt gcaggaagcc acccccctgg gccctgccag ctccctgccc 120 cagagcttcc tgctcaagtg cttagagcaa gtgaggaaga tccagggcga tggcgcagcg 180 ctccaggaga agctgtgtgc cacctacaag ctgtgccacc ccgaggagct ggtgctgctc 240 ggacactctc tgggcatccc ctgggctccc ctgagcagct gccccagcca ggccctgcag 300 ctggcaggct gcttgagcca actccatagc ggccttttcc tctaccaggg gctcctgcag 360 gccctggaag ggatctcccc cgagttgggt cccaccttgg acacactgca gctggacgtc 420 gccgactttg ccaccaccat ctggcagcag atggaagaac tgggaatggc ccctgccctg 480 cagcccaccc agggtgccat gccggccttc gcctctgctt tccagcgccg ggcaggaggg 540 gtcctggttg cctcccatct gcagagcttc ctggaggtgt cgtaccgcgt tctacgccac 600 cttgcccagc cctga 615 <210> 2 <211> 1247 <212> DNA <213> Artificial Sequence <220> <223> hGCSF-WPRE <400> 2 atggctggac ctgccaccca gagccccatg aagctgatgg ccctgcagct gctgctgtgg 60 cacagtgcac tctggacagt gcaggaagcc acccccctgg gccctgccag ctccctgccc 120 cagagcttcc tgctcaagtg cttagagcaa gtgaggaaga tccagggcga tggcgcagcg 180 ctccaggaga agctgtgtgc cacctacaag ctgtgccacc ccgaggagct ggtgctgctc 240 ggacactctc tgggcatccc ctgggctccc ctgagcagct gccccagcca ggccctgcag 300 ctggcaggct gcttgagcca actccatagc ggccttttcc tctaccaggg gctcctgcag 360 gccctggaag ggatctcccc cgagttgggt cccaccttgg acacactgca gctggacgtc 420 gccgactttg ccaccaccat ctggcagcag atggaagaac tgggaatggc ccctgccctg 480 cagcccaccc agggtgccat gccggccttc gcctctgctt tccagcgccg ggcaggaggg 540 gtcctggttg cctcccatct gcagagcttc ctggaggtgt cgtaccgcgt tctacgccac 600 cttgcccagc cctgagccaa gggtaccgag ctcgaattcg atttctgttc ctgttaatca 660 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt 720 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc 780 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc 840 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg 900 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc 960 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg 1020 cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg 1080 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc 1140 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct 1200 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcctg 1247 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 agaagctgtg tgccacctac aagc 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gtacgacacc tccaggaagc tctg 24 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 tgatgccccc atgtttgtga 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 caagaaggga acacgcaggg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 catgaagctg atggccctgc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gctctgcaga tgggaggcaa 20  

Claims (7)

delete An expression retroviral vector characterized in that it is a replication-deficient retrovirus derived from murine leukemia virus containing the recombinant hG-CSF gene of SEQ ID NO: 1. The method of claim 2, The vector is a retroviral vector for expression, characterized in that the plasmid type is pLNC-GCSF (KCTC 11200BP). Animal cell transformed with the retroviral vector according to claim 2. The animal cell of claim 4 is a fetal fibroblast cell, embryonic fibroblast cell of chicken, HeLa cells, mouse NIH3T3 cell or chicken embryo fibroblast cell. Transgenic poultry which can be transformed by the retroviral vector according to claim 2 to express the hG-CSF gene. The method of claim 6, The poultry is a chicken, duck, turkey, geese, quail or pigeon transgenic poultry.

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