• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.62-62
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• 2001

Recently, production of transgenic animal by nuclear transfer has been known as a useful method. The production of cloned offspring derived from nuclear transfer depends upon a variety of factors such as species, donor cells type and cell cycle, and source of recipient ova. Therefore, we attempted a different transgenic methods using follicular granulosa cells (GCs). In general, ovulated GCs undergoes lutenization and transformation in vitro which might defective effects on developmental potential. In order to avoid the GCs transformation in vitro culture system, we introduced a direct injection of retrovirus into the follicles and then collected them mechanically from ovaries of 6-8 week-old ICR mice. Retrovirus vector constructed with pLN $\beta$ EGFP was injected into the follicles. The follicles are cultured in $\alpha$ -MEM supplemented with human FSH, LH and ITS in Costar Transwell dish for 4 days. Survival rate of virus injected follicles was 52.1% (12/23) and expression rate of EGPP gene was 33.3% (4/12). In this study, we found GCs performed transgenesis in our culture system. In addition, the GCs in follicle may be developed in vivo like environment rather than in vitro environment. Thus, the use of GCs as donor cells may be useful in the nuclear transfer for cloning of genetic modification. Therefore, these results suggest that follicular GCs can be transfected by viral vector during folliculogenesis in vitro.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.1
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• pp.27-32
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• 2004

Using MLV (murine leukemia virus)-based retrovirus vectors encapsidated with VSV-G (vesicular stomatitis virus G glycoprotein), we tried to make transgenic chickens carrying the transferred genes in their chromosomes. Twenty one days after virus injection beneath the blastoderms of unincubated chicken embryos (stage Ⅹ, at laying), DNA isolated from the hatched chicks were analyzed by PCR with two sets of primers specific for EGFP (enhanced green fluorescence protein) gene or $Neo^R$ (E. coli neomycin resistant) gene. Among sixty-seven embryos injected with retrovirus, four of them were identified to carry the EGFP genes in their genomes. Remarkably, one transgenic chick showed presence of the retrovirus vector sequences in all organs differentiated from one of endoderm, mesoderm, and ectoderm. Expression of EGFP gene was not detected, however, the stable germ line transmission of transgene was verified in spermatozoa from the founder chicken and 50% of $F_1$ progenies.

• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.193-199
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• 2010

We report here the production of transgenic chickens that can regulate human erythropoietin (hEPO) gene expression. The glycoprotein hormone hEPO is an essential for viability and growth of the erythrocytic progenitors. Retrovirus vector system used in this study has two features including tetracycline-controllable promoter and woodchuck hepatitis virus posttranscriptional regulator element (WPRE). The former is for to reduce the possibility of physiological disturbance due to constitutional and unregulated expression of hEPO gene in the transgenic chicken. The latter is for maximum expression of the foreign gene when we turn-on the gene expression. A replication-defective Moloney murine leukemia virus (MoMLV)-based vectors packaged with vesicular stomatitis virus G glycoprotein (VSV-G) was injected beneath the blastoderm of non-incubated chicken embryos (stage X). Out of 325 injected eggs, 28 chicks hatched after 21 days of incubation and 16 hatched chicks were found to express the hEPO gene delivered by the vector. The biological activity of the recombinant hEPO in transgenic chicken serum was comparable to its commercially available counterpart. The recombinant hEPO in transgenic chicken serum had N- and O-linked carbohydrate simillar to that produced from in vitro cultured cells transformed with hEPO gene.

• Reproductive and Developmental Biology
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• v.37 no.3
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• pp.91-96
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• 2013

Transgenic chickens have been spotlighted as an highly potent bioreactor for their fecundity, short generation time, and eggs associated with mass production of protein. In this study, we generated transgenic chickens exhibiting oviduct specific expression of human growth hormone fused to human transferrin for oral administration. Gene of the modified growth hormone located at downstream ovalbumin promoter (~3.6 kb) was introduced to stage X blastodermal cell employing retrovirus vector system. Several transgenic chickens were successfully generated. However, genomic analyses showed unexpected deletion within the transgene. The modification of the transgene seemed to occur during germ cell formation because the deletion was detected only from the sperm DNA of the G0 founder animal. There was no evidence of deletion in the somatic cell DNA samples of the same chicken. Consequently, same pattern of the deletion was confirmed in both somatic and germ cells of the G1 progeny.

• Korean Journal of Poultry Science
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• v.32 no.4
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• pp.225-229
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• 2005

Microinjection of recombinant retrovirus beneath the blastoderm of non-incubated chicken embryo is now the most widespread method for generating transgenic chickens, but transgenesis rates are very low. So to improve this problem, we first introduced retrovirus vector carrying RSV-GFP gene to an one-cell embryo culture system. To investigate whether retrovirus could work on an one-cell chicken embryo, we microinjected the concentrated retrovirus stocks into the germinal disc of one cell or stage-X chicken embryos. Analysis of reporter gene expression on day 4 embryos showed that GFP expression was observed in the only stage-X chicken embryo but was not in the one-cell embryo group. These results suggest that retrovirus system is the most efficient method to generate transgenic chickens in the stage-X embryo.

• Journal of Microbiology and Biotechnology
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• v.30 no.9
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• pp.1273-1281
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• 2020

Due to the broad host suitability of viral vectors and their high gene delivery capacity, many researchers are focusing on viral vector-mediated gene therapy. Among the retroviruses, foamy viruses have been considered potential gene therapy vectors because of their non-pathogenicity. To date, the prototype foamy virus is the only retrovirus that has a high-resolution structure of intasomes, nucleoprotein complexes formed by integrase, and viral DNA. The integration of viral DNA into the host chromosome is an essential step for viral vector development. This process is mediated by virally encoded integrase, which catalyzes unique chemical reactions. Additionally, recent studies on foamy virus integrase elucidated the catalytic functions of its three distinct domains and their effect on viral pathogenicity. This review focuses on recent advancements in biochemical, structural, and functional studies of foamy virus integrase for gene therapy vector research.

• Journal of Microbiology and Biotechnology
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• v.11 no.2
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• pp.234-241
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• 2001

The C-terminal 393 bp region of the human interleukin-16 (IL-16) gene was cloned and expressed in E. coli along with mammalian cell lines. Recombinant IL-16 expressed from E. coli was 22 kDa on SDS-PAGE and showed 260% of chemoattractant activity at a concentration of $0.1\;{\mu}g/ml$. HeLa, COS, and Neuro-2a cells were transduced by recombinant retrovirus vector pLNC/IL-16/IRES/TK and the intracellular and secreted amounts of IL-16 produced by HeLa/IL-16/TK, COS/IL-16/TK, and Neuro-2a/IL-16/TK cells were determined by enzyme-linked immunosorbent assay (ELISA). HeLa/IL-16/TK $(1{\times}10^5)$ and COS/IL-16/TK $(1{\times}10^5)$ cells secreted 36.1 and 13.3 ng of IL-16 for 48 h, respectively. Forty-nine ng and 86.4 ng of IL-16 remained in the cell lysates of HeLa/IL-16/TK and COS/IL-16/TK. Intracellular and secreted amounts of IL-16 from Neuro-2a/IL-16/TK $(5{\times}10^5)$ cells during 24 h cultivation were 50 ng and 3.3 ng, respectively. Also, HeLa and COS cells wee stably transfected with mammalian expression vector pCRIII/IL-16. Both culture media and cell lysates prepared from HeLa/IL-16 cells and COS/IL-16 cells showed chemoattractant activity ranging from 190% to 460% as compared to the control experiment. Expression of the herpes simplex virus thymidine kinase (HSV0tk) gene in pLNC/IL-16/ IRES/TK bicistronic retroviral expression vector was verified by performing a genciclovir (GCV) sensitivity assay. Finally, IL-16 repressed Tat-transactivated human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR) promoter activity.

• Tuberculosis and Respiratory Diseases
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• v.49 no.3
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• pp.298-309
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• 2000

Background : The antitumor effects of herpes simplex virus thymidine kinase (HSV-tk) and ganciclovir (GCV) strategies for cancer gene therapy have a the following advantages : 1) a direct cytotoxicity to HSV-tk modified cancer cells by GCV 2) a cell death by the local transfer of toxic metabolites from the HSV-tk modified cells to nearby unmodified tumor cells (bystander effect), and 3) in vivo bystander effect such as antitumor-immunity. Retroviral and adenoviral sequences can silence transgene expression in cells and mice. In this study, we investigated the above described advantages of HSV-tk/GCV strategy in Lewis lung cell and mouse lung cancer model using retroviral vector and adenoviral vector. Also, we observed whether the expression of a silenced gene can be reactivated by treating cells with butyrate. Methods : Retrovirus-HSV-tk and adenovirus-HSV-tk vectors were used for the transduction of Lewis lung carcinoma (LLC) cells. The change of HSV-tk expression by butyrate was measured by Western blol The antitumor activities containing bystander effect were observed in vivo (by MTT assay) and in vivo tumor models of various combinations of LLC and LLC-tk. Results : 1. Butyrate induced the enhancement of HSV-tk expression from adenovirally transduced cells but not from retrovirally transduced cells. 2. Both retrovirus-HSV-tk and adenovirus-HSV-tk vectors with GCV treatment were effective for killing of tumor cell in vitro and suppression of LLC tumorigenicity. Bystander effect was responsible for killing of mixture of LLC-tk and LLC in vitro and in vivo-tumorigenicity model. Conclusion : Butyrate could augment adenovirus-mediated HSV -tk gene expression. Cancer gene therapy with HSV-tk suicide gene by retroviral and adenoviral vector seems to be an effective approach for lung cancer therapy.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.23-23
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• 2002

In vitro matured porcine oocytes have very small volume of perivitellinespace(PVS). In these respect, the effects of sucrose and polybrene on the efficiency of gene transfer were investigated. As a gene (hGH) transfer vehicle, Vesicular stomatitis virus glycoprotein pseudotyped retroviral vector (VSV-G) was used. Sucrose treatment have no detrimental effect on the rates of cleavage and following development and induced the enlargement of PVS resulting the efficient introduction of retroviral vector stocks into PVS. (omitted)

• 대한생식의학회:학술대회논문집
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• 1999.11a
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• pp.37.1-37.1
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• 1999