• 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.

• Journal of fish pathology
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• v.30 no.2
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• pp.71-78
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• 2017

The glycoprotein of novirhabdoviruses is known to play a critical role in the determination of host specificity. Viral hemorrhagic septicemia viruses (VHSVs) in different genotypes have different glycoprotein sequences and show different preferences for specific cell lines. In this study, to know whether the glycoprotein is solely responsible for the host cell preference of VHSV, a recombinant VHSV expressing vesicular stomatitis virus (VSV) glycoprotein instead of VHSV IVa glycoprotein (rVHSV-VSV-G) was generated by reverse genetics and inoculated into several fish cell lines, then, cytopathic effect (CPE) and viral growth caused by rVHSV-VSV-G infection were compared with those caused by rVHSV-wild that was previously generated and has the same genomic sequence with wild-type VHSV except a few nucleotides. The plaque numbers of rVHSV-VSV-G were significantly higher in EPC, BF-2 and GF cells than those of rVHSV-wild. However, in HINAE cells (originated from olive flounder), rVHSV-VSV-G titer was significantly lower than rVHSV-wild titer, and both recombinant VHSVs were not grown well in CHSE-214 cells. Although statistical significances were detected in the titers between rVHSV-wild and rVHSV-VSV-G in several cell lines, the cell line-preference order of rVHSV-VSV-G was not different from that of rVHSV-wild. These results suggest that the replacement of VHSV glycoprotein may not completely change host cell preference, and other regions of VHSV might also involve in the determination of host cell preference.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.2
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• pp.163-169
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• 2001

Despite the high potency of the retrovirus vector system in gene transfer, one of the main drawbacks of has been difficulty in preparing highly concentrated virus stock. Numerous efforts to boost the virus titer have ended in unsatisfactory results mainly due to fragile property of retrovirus envelope protein. In this study, to overcome this problem, we constructed our own retrovirus vector system producing vector viruses encapsulated with VSV-G (vesicular stomatitis virus G glycoprotein). Concentration process of the virus stock by ultracentrifuge did not sacrifice the virus infectivity, resulting in more than 108 to 109 CFU (colony forming unit) per ml on most of the target cell lines tested. Application of this high-titer retrovirus vector system was tested on chicken embryos. Injection of virus stock beneath the blastoderms of pre-incubated fertilized eggs resulted in chick embryos expressing E. coli LacZ gene with 100% efficiency. Therefore, our results suggest that it is possible to transfer the foreign gene into chicken embryo using our high-titer retrovirus vector.

• Proceedings of the Zoological Society Korea Conference
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• 2001.04a
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• pp.94.1-94
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• 2001

No Abstract, See Full Text

• Journal of Microbiology and Biotechnology
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• v.27 no.6
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• pp.1098-1105
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• 2017

Vesicular stomatitis virus G glycoprotein (VSV-G) has been widely used for pseudotyping retroviral, lentiviral, and artificial viral vectors. The objective of this study was to establish a potential approach for large-scale production of VSV-G. To this end, VSV-G was cloned with an N-terminal His-tag into Pichia pastoris expression vector pPIC3.5K. Three clones ($Mut^s$) containing the VSV-G expression cassette were identified by PCR. All clones proliferated normally in expansion medium, whereas the proliferation was reduced significantly under induction conditions. VSV-G protein was detected in cell lysates by western blot analysis, and the highest expression level was observed at 96 h post induction. VSV-G could also be obtained from the condition medium of yeast protoplasts. Furthermore, VSV-G could be incorporated into Ad293 cells and was able to induce cell fusion, leading to the transfer of cytoplasmic protein. Finally, VSV-G-mediated DNA transfection was assayed by flow cytometry and luciferase measurement. Incubation of VSV-G lysate with the pGL3-control DNA complex increased the luciferase activity in Ad293 and HeLa cells by about 3-fold. Likewise, incubation of VSV-G lysate with the pCMV-DsRed DNA complex improved the transfection efficiency into Ad293 by 10% and into HeLa cells by about 1-fold. In conclusion, these results demonstrate that VSV-G could be produced from P. pastoris with biofunctionalities, demonstrating that large-scale production of the viral glycoprotein is feasible.

• Biomolecules & Therapeutics
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• v.22 no.2
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• pp.114-121
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• 2014

Refractoriness of acute myeloid leukemia (AML) cells to chemotherapeutics represents a major clinical barrier. Suicide gene therapy for cancer has been attractive but with limited clinical efficacy. In this study, we investigated the potential application of herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) based system to inhibit chemoresistant AML cells. We first generated Ara-C resistant K562 cells and doxorubicin-resistant THP-1 cells. We found that the HSV-TK/GCV anticancer system suppressed drug resistant leukemic cells in culture. Chemoresistant AML cell lines displayed similar sensitivity to HSV-TK/GCV. Moreover, HSV-TK/GCV killing of leukemic cells was augmented to a mild but significant extent by all-trans retinoic acid (ATRA) with concomitant upregulation of Connexin 43, a major component of gap junctions. Interestingly, HSV-TK/GCV killing was enhanced by expression of vesicular stomatitis virus G glycoprotein (VSV-G), a fusogenic membrane protein, which also increased leukemic cell fusion. Co-culture resistant cells expressing HSV-TK and cells stably transduced with VSV-G showed that expression of VSV-G could promote the bystander killing effect of HSV-TK/GCV. Furthermore, combination of HSV-TK/GCV with VSV-G plus ATRA produced more pronounced antileukemia effect. These results suggest that the HSV-TK/GCV system in combination with fusogenic membrane proteins and/or ATRA could provide a strategy to mitigate the chemoresistance of AML.

• 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.

• Proceedings of the Korea Society of Poultry Science Conference
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• 2003.11a
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• pp.95-96
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• 2003

본 연구에서는 VSV-G(vesicular stomatitis virus G glycoprotein)로 포장된 MoMLV(Moloney murine leukemia virus) retrovirus vector system을 이용하여 GFP가 발현되는 형질전환 닭을 생산하고자 하였다. GFP 유전자를 retroviral vector 내의 RSV(Rous sarcoma virus) promoter의 조절 하에 도입한 후, Gp293 세포주에서 virus 형태로 생산하였으며, 이 virus를 초원심분리로 고농축하여 stage X 계란의 배 반엽 층에 주입하여 GFP가 발현되는 형질전환 닭을 생산하였다. 생산된 닭에서의 GFP의 발현은 epifluorescence stereomicroscope를 이용하여 확인하였다. 이 방법은 기존의 여러 형질전환 가금 방법에 비하여 기술적인 용이성과 경제성을 가지므로(Muramatsu, Park and Okumura 1998), 매우 효율적이고 주목할 만한 형질전환 가금 생산 방법으로 사료된다. 형질전환 가금의 생산에서 retrovirus vector를 이용하는 방법은 다양한 종류의 표적세포에 대하여 retrovirus 고유의 감염성에 의한 외래 유전자의 전이가 용이하고, 전이된 유전자가 진정염색질 영역 내로 선택적으로 도입될 수 있으며 유전적으로 안정성을 나타내므로 매우 효과적인 방법이다. 그러나 가금에서는 초기 배 발달에 의한 급격한 세포의 수적 증가로 인해 고감염성 virus의 획득이 요구되므로, 본 연구에서는 virus의 농축에 있어 보다 안정적이고 숙주 범위에 있어서 pantropic한 VSV-G에 기반을 둔 retrovirus vector system을 확립하였다. 이 system은 기존의 형질전환 닭의 생산방법에 비해 외래 유전자의 전이에 있어서 매우 효과적인 것으로 확인되었으며, 또한 여러 유용한 생리활성물질을 분비하는 형질전환 동물의 생산에 있어서 상당한 기여를 할 것으로 사료된다.

• Reproductive and Developmental Biology
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• v.32 no.3
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• pp.159-166
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• 2008

We report here the generation of transgenic chickens that produce human Thrombopoietin (hTPO) using replication-defective Moloney murine leukemia virus (MoMLV)-based vectors packaged with vesicular stomatitis virus G glycoprotein (VSV-G). For the retrovirus vectors, we used hCMV (human Cytomegalovirus) internal promoter to drive the hTPO gene. After confirming the expression of the hTPO gene in various target cells, the concentrated solution of recombinant retrovirus was injected beneath the blastoderm of non-incubated chicken embryos (stage X). The biological activity of the recombinant hTPO in target cell was significantly higher than its commercially available counterpart. Out of 132 injected eggs, 11 chicks hatched after 21 days of incubation and 4 hatched chicks were found to express vector-encoded hTPO gene. However, 3 out of the 4 transgenics died within one month of hatching. The major significance of this study is that it is one of the very few successful reports on the production of transgenic chickens as bioreactors aiming mass production of commercially valuable and biological active human cytokine proteins.

• 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)