• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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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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• 제17권1호
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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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• 제34권3호
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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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• 제37권3호
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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.

• 한국가금학회지
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• 제32권4호
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• pp.225-229
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• 2005

현재 가장 활발하게 진행되고 있는 형질전환 자 생산 연구방법은 배반엽단계 수정란에 농축한 virus를 주입하여 모자이크 형태의 $G_0$ 형질 전환체를 생산하고 이들을 이용하여 $G_1$ 형질전환 후대를 생산하는 방법이 가장 보편적으로 이용되고 있다. 상기의 연구방법은 완전한 형질 전환체를 획득하기 위해서는 수천수의 $G_1$을 생산하고 각각 유전분석을 수행하는 문제점을 가지고 있다. 이러한 문제점을 개선하고자 다음의 연구를 계획하고 수행하였다. 20nL의 농축된 GFP retrovirus를 1세포기 수정란에 주입하고, 주입한 유전자의 발현율과 수정란의 생존율을 배양 4일차 수정란에서 GFP의 발현과 배자의 생존 여부로 판정하였다. 연구결과는 배양 4일차 수정란의 생존율은 기존의 naked DNA 미세주입방법에 비해 다소 낮은 것으로 나타났으나 유의성은 없었다. 1세포기 수정란은 배반엽 단계 수정란과 달리 주입한virus의 유전자를 발현하지 않는 것으로 관찰되었다. 연구결과는 배반엽단계 수정란에 virus 미세주입 방법이 형질전환 닭 생산에 가장 효율적인 방법임 보여주고 있다.

• Journal of Microbiology and Biotechnology
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• 제30권9호
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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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• 제11권2호
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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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• 제49권3호
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• pp.298-309
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• 2000

연구배경 : 암 유전자치료에서 각광받고 있는 HSV-tk/GCV 전략의 항암효과에는 다음과 같은 장점들이 거론되고 있다 : 1) GCV 처리에 의한 암세포 직접살상효과 2) HSV-tk 이입된 세포에 의해서 HSV-tk 이입되지 않은 주변세포를 살상하는 bystander effect 3) 생체 내 bystander eff ect로 알려 진 anti-tumor immunity. Retrovirus와 adenovirus sequence를 이용할 경우 몇몇 세포주와 마우스에서 이들이 목적유전자의 발현을 억제할 수 있다는 것이 보고되고 있다. 본 연구에서는 retroviral나 adenoviral vector로 HSV-tk 유전자를 이입한 Lewis 폐암세포주와 폐암 마우스 모델을 통하여 HSV-tk/GCV 전략의 장점을 조사하였고 이 viral vector들 사이의 차이를 비교 조사하였다. 또한 Lewis 폐암세포주에서 butyrate를 처리한 후 HSV-tk 유전자의 발현증가를 관찰하였다. 방법 : Lewis retroviral vector와 adenoviral vector로 HSV-tk 유전자를 이입한 후 butyrate로 HSV-tk 유전자의 발현을 유도하고 Western blotting수행하여 분석하였다. 생체 외에서 HSV-tk/GCV에 의한 세포살상효과를 MTT 검사로 수행하였고 생체 내에서 LLC 나 HSV-tk 이입된 LLC 세포주를 이식하여 종양소멸 및 bystander effect를 조사 하였다. 결과 : 1. Butyrate로 HSV-tk adenovirus로 이입된 LLC에서 증가한 반면 retrovirus로 이입된 LLC에서는 증가하지 않았다. 2. 생체 외 그리고 생체 내에서 viral vector로 HSV-tk를 이입한 종양세포에 GCV 투여하는 것은 종양 세포의 살상에 효과적이었으며 LLC와 LLC-tk 세포주를 혼합한 실험에서 bystander effect도 종양세포의 성장을 억제하는 것으로 관찰되었다. 결론 : 향후 생체 외 그리고 생체 내 실험에서 adenoviral vector를 이용한 유전자 전달에 butyrate를 함께 사용하면 유전자발현을 증진시킬 것으로 사료되며 자살 유전자인 HSV-tk을 종양에 이입하여 GCV을 처리 하는 치료가 폐암유전자치료에 효과가 있을 것으로 생각된다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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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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• 대한불임학회 1999년도 제38차 추계 학술대회
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• pp.37.1-37.1
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• 1999