• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.197-202
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• 2004

Human PTH (parathyroid hormone) is known to be efficacious for curing osteoporesis. In this study, we attempted to construct genetically modified porcine cell lines that can ultimately be used for donor cells in nuclear transfer-mediated transgenesis. By using retrovirus vectors carrying tetracycline-regulatory expression system and WPRE (woodchuck hepatitis virus posttranscriptional regulatory element) sequence, we could control PTH expression with tetracycline and boost the promoter activity. Considering that low or constitutive expression of the transgene has been one of major problems that needs to be solved in transgenic animal studies, our results could be helpful in successful production of transgenic pigs as bioreactors.

• Proceedings of the Korea Society of Poultry Science Conference
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• 2005.11a
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• pp.70-71
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• 2005

본 연구는 1세포기 닭 수정란에 retrovirus vector (RSV-GFP)를 도입하여 외래유전자의 핵 전이 효율을 높이고자 하였다. 실험은 polybrene과 retrovirus 혼합물을 1세포기 또는 배반엽 단계의 수정란 세포질에 미세주입하고 배양 3 또는 4일차에 GFP의 발현 양상들을관찰하였다. 실험의 결과는 배반엽 수정란에서 GFP발현을 관찰할 수 있었으나, 1세포기 수정란에서는 GFP의 발현을 관찰할 수 없었다. 연구결과는 형질전환닭 생산에 있어서 가장 효율적인 방법은 배반엽 단계에 retrovirus를 미세주입하는 방법임을 보여주고 있다.

• Journal of Microbiology and Biotechnology
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• v.15 no.2
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• pp.451-454
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• 2005

Introduction of foreign genes into brain cells, such as neurons and astrocytes, is a powerful approach to study the gene function and regulation in the neuroscience field. Calcium phosphate precipitates have been shown to cause cytotoxicity in some mammalian cells and brain cells, thus leading to low transfection efficiency. Here, we describe a retrovirus-mediated gene delivery method to transduce foreign genes into brain cells. In an attempt to achieve higher gene delivery efficiency in these cells, we made several changes to the original method, including (1) use of a new packaging cell line, Phoenix ampho cells, (2) transfection of pMX retroviral DNA, (3) inclusion of 25 mM chloroquine in the transduction, and (4) 3- 5 h incubation of retroviruses with target cells. The results showed that the modified protocol resulted in a range of 40- 60% gene delivery efficiency in neurons and astrocytes. Furthermore, these results suggest the potential of the retrovirus-mediated gene delivery protocol being modified and adapted for other transfection-refractory cell lines and primary cells.

• Korean Journal of Animal Reproduction
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• v.20 no.3
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• pp.333-341
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• 1996

One of the biggest problems involved in transgenic animal production is lack of appropriate market genes. To overcome this problem, we tested whether the genes of SEAP (secreted alkaline phosphatase) and GFP (green fluorescence protein) on our retrovirus vectors can be applicable to the transgenic animal production. The main advantage of these marker genes over other generally mainpulation can be selected without sacrificing viability. The results obtained in this study are summarized as follows: 1. Removal of zona pellucida from the mouse zygotes did not affect embryo developments to blastocysts. 2. Co-culture of zona-free embryos with virus-producing cells for 6 hours also did not affect embryo developments to blastocysts. 3. Among 58 blastocysts developed from the zona-free zygotes co-cultured with the virus-producing cells, SEAP expression was observed from the 6 blastocysts. 4. Expression of the GFP gene was detected from the virus- producing cells but no embryo expressing the gene was counted among 50 blastocysts developed from the zona-free zygotes co-cultured with the virus-producing cells.

• Korean Journal of Animal Reproduction
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• v.27 no.3
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• pp.259-268
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• 2003

Lactadherin (formerly known as BA46), a major glycoprotein of the human milk fat globule membrane, is abundant in human breast milk and breast carcinoma cells and is known to prevent symptomatic rotavirus infections. In this study, we tried to transfer the human lactadherin gene to the Chinese Hamster Ovary (CHO) cells using retrovirus vector system and tested inducible expression of the gene under the tetracycline-controllable promoter. At first, tetracycline-mediated inducibility was tested using E.coli LacZ marker gene. NIH3T3 cells co-infected with RevTet-On and RevTRE-LacZ retrovirus vectors showed that the cells responded to doxycycline (a derivative of tetracycline) in a dose-dependent manner, and prominent induction of the lacZ gene expression was observed from 1 $\mu\textrm{g}$/ml of doxycycline concentration. Based on the results of the pilot experiment, inductional expression of the human lactadherin gene was conducted using RevTet-On and RevTRE-Ltd retrovirus vectors. Analysis with the RT-PCR demonstrated successful inductional expression of the lactadherin gene in the Chinese Hamster Ovary (CHO) cells. Considering that constitutive overexpression of the exogenous genes in the target cells causes serious physiological imbalance, the results obtained in this study will be very useful especially in the studies of gene therapy and transgenic animal production.

• Reproductive and Developmental Biology
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• v.32 no.1
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• pp.9-14
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• 2008

Efficient gene transfer into hematopoietic stem cells is a great tool for gene therapy of hematopoietic disease. Retrovirus have been extensively used for gene delivery and gene therapy. However, current in vitro gene transfer has some obstacles suck as induction of differentiation loss of self-renewal capacity, and down-regulation of homing efficiency for in vitro hematopoietic stem cells transplantation. To overcome these problems, we developed efficient in vitro retroviral transfer technique by direct intra-bone marrow injection (IBM). We identified effective retrovirus gene transfer in bone marrow hematopoietic cells in vitro. Two weeks after retrovirus transfer via IBM injection, we observed stable EGFP gene expression in bone marrow, lymph node, spleen, and liver cells. In addition, $6.4{\pm}2.7%$ of hematopoietic stem/progenitor cells were expressed EGFP transgene from flow cytometry analysis. Our results demonstrate that in vitro retrovirus gene transfer via IBM injection can provide a viable alternative to current or moo gene transfer approach.

• Journal of fish pathology
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• v.22 no.3
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• pp.375-382
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• 2009

Integration and expression of a target gene into chromosomal genomes of host cell by retrovirus mediated gene transfer system usually require complicate and laborious procedures. In the present study, we investigate a simple method to integrate a target gene into genome of BF-2 cells using ultraviolet (UV)-inactivated snakehead retrovirus (SnRV), a fish retrovirus. First of all, an optimization of transfection condition was determined with BF-2 cells using Lipofectamine 2000 and Transome. Using 0.5 $\mu\ell$ Lipofectamine 2000 resulted in 33.8, 40.6 and 40.2% of transfection efficacy with high survival rate (minimum 80%) in 0.5, 1 and 2 $\mu{g}$ DNA, respectively, and those of Transome were all less than 5%. It was confirmed that UV-treatment for 5 min was enough to inactivate infectivity of SnRV. Next, a cassette composed of GFP (green fluorescent protein) gene flanked by LTR (long terminal repeats) sequences derived from SnRV was constructed and transfected into BF-2 cells followed by treatment with UV-inactivated SnRV for optimization of integration and expression of the cassette gene. As the results, the fluorescence was expressed in BF-2 cells treated with UV-inactivated SnRV 3 and 5 times, while there was no expression in BF-2 cells with once and non treatment. Accordingly, it was confirmed that GFP gene was integrated into chromosomal genome of BF-2 cells with UV-inactivated SnRV.

• Journal of Life Science
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• v.25 no.4
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• pp.481-485
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• 2015

Retroviruses genes have been inserted into the human genome for millions of years. These retroviruses are now inactive due to mutations such as deletions or nonsense mutations. After mutation, retroviruses eventually became fixed in the genome in their endogenous forms and existed as traces of ancient viruses. These retroviruses are called endogenous retroviruses (ERVs), with the human form known as human endogenous retrovirus. HERV cannot become a fully active virus, but a number of viral proteins or even virus particles are expressed under various conditions. Compared to endogenous retroviruses, some exogenous retroviruses are still infectious and can threaten human life. Among these, human immunodeficiency virus (HIV) is one of the most well-known and best-studied. Recent studies have shown some elements of HERV were activated by HIV infection and interact with HIV-derived proteins. In addition, many studies have attempted to use HERV as vaccination against HIV infection. This review will describe the regulation and interaction between HERV and HIV infection and mention the development of vaccines and therapeutic agents against HIV infection by using HERV elements.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.239-239
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• 2004

본 연구에서는 효과적인 유전자를 발현시킬 수 있는 retrovirus vector system을 구축하기 위하여 네 가지 promoter를 비교하였다. 일반적으로 사용되고 있는 RSV (Rous sarcoma virus), UbC (Ubiquitin), β-actin, CMV(cytomegalovirus) promoter 하에 GFP (green fluorescent protein)를 표지유전자로 사용하였다. 또한 WPRE (Woodchuck hepatitis virus Posttranscriptional Regulatory Element) 서열을 도입하여 각각의 promoter 하에서 GFP 유전자의 발현 증가 여부를 검정하였다. (중략)

• Reproductive and Developmental Biology
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• v.29 no.1
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• pp.49-55
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• 2005

We cloned cDNA of the PGH(porcine growth hormone) gene and constructed retrovirus vector designed to express PGH gene under the regulation of CMV (cytomegalovirus) promoter. To maximize the expression, WPRE(woodchuck hepatitis virus posttranscriptional regulatory element) sequence was placed at the downstream of the PGH gene. After infection with recombinant viruses, approximately 1×10/sup 6/ PFF(porcine fetal fibroblast) cells released PGH protein into the media as much as 1,400 ng. In a subsequent experiment, a modifications of the retrovirus vector was made to express the PGH gene in a teracycline-inducible manner. In PFF cells carrying these viral vector sequences, addition of doxycycline to the media resulted in 2∼6 fold increase in PGH synthesis. In the modified retrovirus vectors, the WPRE sequence also played a role in boosting the effect of the tetracycline induction. This result indicates that our tetracycline-inducible expression system might be a promising candidate in alleviating the complicate physiological problems caused by constitutive expression of the exogenous genes in the transgenic animals.