• BMB Reports
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• v.30 no.4
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• pp.269-273
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• 1997

Heptocvte-specific expression induced by Hepatitis B virus (HBV) enhancer 2-core gene promoter was examined in various hepatocyte and non-hepatocyte cell lines. using non-viral and retroviral vector systems in which chloramphenicol acetyltransferase (CAT) is used as a reporter. The non-viral plasmid containing the HBV enhancer 2-core promoter exhibited 22 and 66% of CAT activities in hepatoma cell lines. HepG2 and Hep3B, respectively when compared with CAT activity expressed by CMV promoter. The CAT activities, however. were found to be marginal in other tested hepatoma cell lines as well as mouse primary hepatocytes and non-hepatocytes. The HBV enhancer 2 located upstream the CMV promoter did not affect the CMV promoter activity nor provided hepatocyte-specific expression. Transfection of retroviral plasmid DNA containing the HBV enhancer 2-core promoter as an internal promoter exhibited high and specific CAT expression in HepG2 and Hep3B cell lines but the activity value was 5 to 10 fold lower than the non-viral plasmid with identical promoter. These results suggest that the usage of HBV enhancer 2-core promoter for liver specific expression is limited to certain vectors and hepatocyte cell lines.

• BMB Reports
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• v.55 no.12
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• pp.615-620
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• 2022

The murine leukemia virus-based semi-retroviral replicating vectors (MuLV-based sRRV) had been developed to improve safety and transgene capacity for cancer gene therapy. However, despite the apparent advantages of the sRRV, improvements in the in vivo transduction efficiency are still required to deliver therapeutic genes efficiently for clinical use. In this study, we established a gibbon ape leukemia virus (GaLV) envelope-pseudotyped semi-replication-competent retrovirus vector system (spRRV) which is composed of two transcomplementing replication-defective retroviral vectors termed MuLV-Gag-Pol and GaLV-Env. We found that the spRRV shows considerable improvement in efficiencies of gene transfer and spreading in both human glioblastoma cells and pre-established human glioblastoma mouse model compared with an sRRV system. When treated with ganciclovir after intratumoral injection of each vector system into pre-established U-87 MG glioblastomas, the group of mice injected with spRRV expressing the herpes simplex virus type 1-thymidine kinase (HSV1-tk) gene showed a survival rate of 100% for more than 150 days, but all control groups of mice (HSV1-tk/PBS-treated and GFP/GCV-treated groups) died within 45 days after tumor injection. In conclusion, these findings sug-gest that intratumoral delivery of the HSV1-tk gene by the spRRV system is worthy of development in clinical trials for the treatment of malignant solid tumors.

• Proceedings of the Microbiological Society of Korea Conference
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• 2002.10a
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• pp.125-128
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• 2002

• Proceedings of the PSK Conference
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• 2000.10a
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• pp.111-112
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• 2000

• Journal of Microbiology and Biotechnology
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• v.21 no.4
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• pp.387-390
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• 2011

Nude mice (BALB/c) were grafted with human 293 cells and PERV (porcine endogenous retrovirus)-IRES-EGFP (a packageable retroviral vector plasmid containing an internal ribosome entry site-enhanced green fluorescent protein)-producing pig PK15 cells in order to determine whether the pig cells could transmit PERV-IRES-EGFP to mice and human 293 cells in vivo. None of the transplanted human 293 cell lines were infected by PERV, but PCR analysis identified PERV-B provirus integration into both the heart and salivary gland of the inoculated nude mice. Our data indicate that hearts and salivary glands can be used to identify PERV-B receptors.

• Korean Journal of Animal Reproduction
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• v.25 no.2
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• pp.171-180
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• 2001

Compared to other gene transfer system, the advantages of retrovirus-mediated gene transfer are technical ease, efficient expression and genetic stability. Despite the high potency of the retrovirus vector system in gene transfer, one of the drawbacks is a difficulty in concentration of virus stock. To overcome this problem, we tested a new retrovirus vector system producing the progeny retrovirus particles encapsidated with VSV-G (vesicular stomatitis virus G glycoprotein). The infectivity of this virus was not sacrificed by ultracentrifugal concentration and the host cell range extended from all mammalian to fish embryos. Virus titer after 1,000 x concentration was more than 10$^{8}$ CFU/ $m\ell$ on most of the target cell lines. We applied this pantropic viruses in transgenic chicken production by injecting the concentrated (100$\times$) stock into subgerminal cavity of stage X chicken embryos. The survival rate of chicken embryos after injection was about 20% and gene integration rate in surviving embryos was scored almost 100%. Analyses of RT-PCR and fluorescence microscopy, however, showed no evidence of the transgene expression.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.49-49
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• 2003

본 연구에서는 vesicular stomatitis virus G glycoprotein (VSV-G)를 envelope로 가지는 pantropic retrovirus vector system을 이용하여 재조합 human FSH 유전자가 전이된 형질전환 닭을 생산하고자 하였다. Human FSH $\alpha$ 및 $\beta$ 유전자와 CTP linker는 human pituitary gland cDNA library에서 RT-PCR 방법을 이용하여 cloning하였으며, 각각의 fragment는 FSH$\beta$-CTP-FSH$\alpha$ 순서의 단일사슬로 연결하였다. 연결된 FSH$\beta$-CTP-FSH$\alpha$는 retroviral vector 내의 $\beta$-actin promoter의 조절 하에 도입한 후, PT67 packaging cell line에 transfection하여 virus를 생산하였으며 생산된 virus는 pantropic한 virus producing cell인 GP293에 infection하여 FSH 유전자가 도입된 virus를 생산하였다. FSH 유전자의 발현을 in vitro에서 확인하기 위하여 CHO (chinese hamster ovary) 세포에 virus를 감염시킨 후, 세포의 배양액을 취하여 electrochemilumine-scence immunoassay 방법으로 정량하였다. In vitro에서 전이 후 발현이 확인된 FSH 외래유전자의 retroviral vector virus를 초원심분리로 고농축하여 stageX의 계란의 배반엽 층에 주입하였으며, 그 결과 18%의 부화율과 91%의 부화한 닭의 유전자 전이율을 확인할 수 있었다. 전이된 유전자의 확인은 FSH$\beta$와 Neo 유전자에 대한 primer를 이용한 RT-PCR의 방법을 이용하였다. In vitro에서와는 달리 in vivo에서는 FSH 유전자의 전이는 확인되었으나 발현을 확인하지는 못하였는데, 이는 적은 수의 실험군이 형질전환율에 비해 상대적이지 못하였거나, 외래 유전자인 FSH의 발현에 의한 생리적인 부작용이 유발되어 해당개체가 부화되지 못한 것으로 추정된다. 본 문제점을 해결하기 위하여 실험군의 수를 늘리고 외래 유전자에 대한 controllable expression system이 보완될 필요성이 요구되며, 이러한 점이 해결된다면 높은 유전자 전이율에 기인하여 retrovirus를 이용한 형질전환 방법은 형질전환 가금의 생산에 있어서 매우 효율적이고 주목할 만한 방법으로 사료된다.

• Proceedings of the PSK Conference
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• 2001.10a
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• pp.215.2-216
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• 2001

• Korean Journal of Animal Reproduction
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• v.26 no.2
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• pp.153-163
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• 2002

In vitro matured porcine oocytes have very small volume of perivitellinspace (PVS). In these respect, the effect of sucrose and polybrene on the efficiency of gene transfer was investigated. As a gene (hGH) transfer vehicle, vesicular stomatitis virus glycoprotein pseudotyped retroviral vector (VSV-G) was used. Sucrose treatment has no detrimental effect on the rates of cleavage and resulted in the enlargement of PVS for the efficient introduction of retroviral vector stocks. Introduction rates of retrovirus in 0.5, 1, 2, 3 % sucrose treatment group were higher than that of the non-treatment group (39.3, 43.3, 35.7, 40.7 % vs. 8.3 %), respectively. In addition, we observed that sucrose pretreatment during injection procedure significantly reduce the frequency of polyspermy. In general, polybrene is a polycation essential for retrovirus transduction. The groups with the addition of 0.5, 5, 50$\mu\textrm{g}$/$m\ell$ polybrene exhibited a significant effect on gene transfer compared to that of the non-addition group (56.5, 50.0, 57.1 % vs. 34.6 %), respectively But, when the oocytes were co-injected with retrovirus and 50$\mu\textrm{g}$/$m\ell$ polybrene, the rates of cleavage and blastocyst development were 43.3 and 4.6%, respectively. This rates were lower than those of the non-addition group (70.0 and 17.3 %). In conclusion, sucrose pretreatment have increased efficiency of retroviral mediated gene transfer in porcine oocytes with no damage on in vitro fertilization and embryo development. In addition, sucrose pretreatment was beneficial in polyspermy inhibition. Presence of polybrene during microinjection showed a beneficial effect on the gene transfer in porcine oocytes, in low concentration. And these results will provide an useful tool for production of transgenic pigs by retroviral mediated gene transfer.

• The Journal of Korean Society of Virology
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• v.26 no.2
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• pp.141-150
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• 1996

본 연구에서는 $CD4^+$ T 세포를 일회성으로 감염시킬 수 있고 $CD4^+$ T 세포에 HIV-1 envelope 유전자를 전달할 수 있는 바이러스 입자를 생산하는 HIV-1 complementation system을 개발하였고 이 system을 이용하여 $CD4^+$ T 세포에 선택적으로 HIV-1 envelope 당단백질을 발현시켰다. 이 system은 Gag/Gag-Pol expressor와 Env expressor로 구성되어있다. Gag/Gag-Pol expressor는 바이러스 입자 생산에 필요한 구조단백질과 기능단백질을 발현시키지만 packaging signal이 결핍되어 바이러스 입자로 유전자가 들어가지 못하도록 제조되었다. Env expressor는 Tat, Rev와 envelope 당단백질을 발현시키고 packaging signal을 갖고 있어 바이러스 입자로 envelope 유전자가 들어가도록 제조되었다. Gag/Gag-Pol expressor로부터 Gag와 Gag-Pol의 발현은 Rev 단백질을 요구하였고 Env expressor로부터 Rev 단백질 이 제공될 때 Gag와 Gag-Pol 단백질은 효율적으로 발현되었다. Gag/Gag-Pol과 Env expressor로 cotransfection된 COS-1 세포에서 $CD4^+$ T 세포를 일회성으로 감염시킬 수 있는 바이러스 입자가 생산되었다. 생산된 바이러스 입자는 $CD4^+$ T 세포에 HIV-1 envelope 유전자를 전달하여 envelope 당단백질을 발현시켰고 복제 가능한 자손 바이러스의 생성을 유도하지 못하였다. 본 연구에서 개발된 방법은 $CD4^+$ T 세포에서 envelope 당단백질의 기능을 분석하고 관심 있는 유전자를 $CD4^+$ T 세포에 전달하는 바이러스 입자의 생산에 이용할 수 있다.