• Animal cells and systems
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• v.6 no.1
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• pp.81-84
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• 2002

SINE-R retroposons have been derived from human endogenous retrovirus HERV-K family and found to be hominoid specific. Both SINE-R retroposons and HERV_K family are potentially capable of affecting the expression of closely located genes. Using the genomic DNA from patients with schizophrenia, we identified 26 SINE-R retroposons and analyzed them with the sequences derived from the hominoid primates. The SINE-R retroposons from schizophrenia showed 89.7-96.6％ sequence similarities with the sequence of the schizo-cDNA clone that derived from postmortem tissue from the frontal cortex of an individual suffering from schizophrenial. Phylogenetic analysis using the neighbor-joining method revealed that the new SINE-R retroposons in schizophrenia have proliferated independently during hominid evolution. Such retroposons have great relevance to genomic change connected to human diseases. The data suggest that new SINE-R retroposons identified in schizophrenia deserve further investigation as potential leads on the understanding of neuropsychiatric diseases.

• Proceedings of the PSK Conference
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• 2002.04a
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• pp.172.2-172.2
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• 2002

• 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 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.

• KSBB Journal
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• v.14 no.4
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• pp.396-402
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• 1999

To verify enchancing effect of polycation of polycation on the retroviral infecivity, we directly measured the binding affinity of retroviruses to the target cells in the presence or in the absence of polybrene with R18 fluorescence assay and examined the effect of the polymers on the relationship between the host cell and the retroviral infecity. There was no difference in the effect of the types of charge of the polymer on the binding affinity. However, polycations, in general, show effect on the retrovirus infecity. This results suggest that the enhancing effect of polybrene and other polycations on the infecity is not due to the binding step but due to the post-binding steps, especially the internalization step. With the result of the internalization of FITC-labeled poly-L-lysine into the host cells, it is suggested that the uptake of polycations into the host cells would play a crucial role in the intermalization of retroviruses.

• Korean Journal of Microbiology
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• v.42 no.3
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• pp.230-234
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• 2006

To study the mechanism of syncytium formation, novel syncytia-inducing ecotropic murine retrovirus was used. Our previous result showed that amino acid substitutions at the RBD (receptor binding domain) of envelope glycoprotein contribute to syncytium formation. In this study, we have investigated if this fusion phenomenon could occur with retroviral vectors pseudotyped with the novel syncytia-inducing ecotropic murine leukemia virus Env. We have found that these vectors were not able to mediate virus-to-cell fusion in M. dunni murine cell lines. These findings indicate that syncytia-inducing ecotropic murine leukemia virus is capable of generating syncytia during its replication. There was also no correlation between the level of ecotropic murine leukemia virus receptor (mCAT-1) and the fusogenic effect.

• BMB Reports
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• v.54 no.7
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• pp.368-373
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• 2021

The vertebrate genome contains an endogenous retrovirus that has been inherited from the past millions of years. Although approximately 8% of human chromosomal DNA consists of sequences derived from human endogenous retrovirus (HERV) fragments, most of the HERVs are currently inactive and noninfectious due to recombination, deletions, and mutations after insertion into the host genome. Several studies suggested that Human endogenous retroviruses (HERVs) factors are significantly related to certain cancers. However, only limited studies have been conducted to analyze the expression of HERV derived elements at protein levels in certain cancers. Herein, we analyzed the expression profiles of HERV-K envelope (Env) and HERV-R Env proteins in eleven different kinds of cancer tissues. Furthermore, the expression patterns of both protein and correlation with various clinical data in each tissue were analyzed. The expressions of both HERV-K Env and HERV-R Env protein were identified to be significantly high in most of the tumors compared with normal surrounding tissues. Correlations between HERV Env expressions and clinical investigations varied depending on the HERV types and cancers. Overall expression patterns of HERV-K Env and HERV-R Env proteins were different in every individual but a similar pattern of expressions was observed in the same individual. These results demonstrate the expression profiles of HERV-K and HERV-R Env proteins in various cancer tissues and provide a good reference for the association of endogenous retroviral Env proteins in the progression of various cancers. Furthermore, the results elucidate the relationship between HERV-Env expression and the clinical significance of certain cancers.

• Tuberculosis and Respiratory Diseases
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• v.46 no.2
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• pp.229-240
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• 1999

Background: The impact of the immune response on cancer gene therapy using viral vectors to deliver a "suicide gene" is currently unclear. A vigrous immune response targeted at viral proteins or transgene may enhance the efficacy of tumor destruction and even augment responses to tumor antigens. These responses may involve the release of cytokines and stimulation of tumor specific cytotoxic T-lymphocytes that enhance therapeutic efficacy. On the other hand, a vigorous rapid cellular immune response may destroy cells expressing the therapeutic gene and attenuate the response to therapy. Furthermore, development of neutralizing antibody responses may prevent readministration of virus, a potentially significant limitation. Evaluating the significance of these limitations in animal models and developing solutions are therefore of obvious importance. Methods: After retroviral transduction of mouse mesothelioma cell line(AB12) with Herpes Simplex Virus thymidine kinase (HSVtk) gene in vitro, subcutaneous flank tumors were established. To study the effect of intact immune system on efficacy of tumor erradication, the ability of the HSVtk/ganciclovir system to inhibit tumor growth was compared among normal Balb/c mice, immunodeficient Balb/c-nude and SCID mice, and Balb/c mice immunosuppressed with cyclosporin. Results: Ganciclovir treatment resulted in greater inhibition of tumor growth in Balb/c mice compared with immunodeficient Balb/c-nude mice and SCID mice(in immunodeficient mice, there were no growth inhibition by ganciclovir treatment). Ganciclovir treatment resulted in greater inhibition of tumor growth in noncyclosporin (CSA) treated Balb/c mice compared with CSA treated Balb/c mice. On day 8, mean ganciclovir-treated tumor volume were 65% of control tumor volume in Balb/c mice versus 77% control tumor volume in CSA-treated Balb/c mice. This effect was still evident during therapy (day 11 and 13). On day 13, non-CSA treated tumor volume was 35% of control tumor volume versus 60% of control tumor volume in CSA treated Balb/c mice. Duration of expression of HSVtk was not affected by the immunosuppression with CSA. Conclusion: These results indicate that the immune responses against retrovirally transduced cells enhance the efficacy of the HSVtk/ganciclovir system. These findings have important implications for clinical trials using currently available retrovirus vectors as well as for future vector design.

• Reproductive and Developmental Biology
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• v.31 no.3
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• pp.161-167
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• 2007

In this study, we constructed and tested retrovirus vectors designed to express the human thrombopoietin gene under the control of the tetracycline-inducible promoters. To increase the hTPO gene expression at him-on state, WPRE sequence was also introduced into retrovirus vector at downstream region of either the hTPO gene or the sequence encoding reverse tetracycline-controlled transactivator (rtTA). Primary culture cells (PFF, porcine fetal fibroblast; CEF, chicken embryonic fibroblast) infected with the recombinant retrovirus were cultured in the medium supplemented with or without doxycycline for 48hr, and induction efficiency was measured by comparing the hTPO gene expression level using RT-PCR, western blot and ELISA. Higher hPTO expression and tighter expression control were observed from the vector in which the WPRE sequence was placed at downstream of the hTPO (in CEF) or rtTA(in PFF) gene. This resulting tetracycline inducible vector system may be helpful in solving serious physiological disturbance problems which have been a major obstacle in successful production of transgenic animals.