• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2003년도 제39회 학술심포지움
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• pp.99-99
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• 2003

• 한국동물학회:학술대회논문집
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• 한국동물학회 2003년도 한국생물과학협회 학술발표대회
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• pp.198.3-198
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• 2003

No Abstract, See Full Text

• Journal of Microbiology and Biotechnology
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• 제18권3호
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• pp.585-590
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• 2008

Human tropic Porcine Endogenous Retroviruses (PERVs) are the major concern in zoonosis for xenotransplantation because PERVs cannot be eliminated by specific pathogen-free breeding. Recently, a PERV A/C recombinant with PERV-C bearing PERV-A gp70 showed a higher infectivity (approximately 500-fold) to human cells than PERV-A. Additionally, the chance of recombination between PERVs and HERVs is frequently stated as another risk of xenografting. Overcoming zoonotic barriers in xenotransplantation is more complicated by recombination. To achieve successful xenotransplantation, studies on the recombination in PERVs are important. Here, we cloned and sequenced proviral PERV env sequences from pig gDNAs to analyze natural recombination. The envelope is the most important element in retroviruses as a pivotal determinant of host tropisms. As a result, a total of 164 PERV envelope genes were cloned from pigs (four conventional pigs and two miniature pigs). Distribution analysis and recombination analysis of PERVs were performed. Among them, five A/B recombinant clones were identified. Based on our analysis, we determined the minimum natural recombination frequency among PERVs to be 3%. Although a functional recombinant envelope clone was not found, our data evidently show that the recombination event among PERVs may occur naturally in pigs with a rather high possibility.

• BMB Reports
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• 제34권2호
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• pp.176-181
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• 2001

Using a retrovirus, foreign genes can be introduced into mammalian cells. The purpose of this study is to produce a retrovirus that can make the infected cells express two genes; the human multidrug resistance gene (MDR1) and the HLA-B7 gene, which is one of the major human histocompatibility complex (MHC) class I genes. For the expression of these genes, the internal ribosome entry site (IRES) was used, which was derived from the encephalomyocarditis (EMC) virus. In order to produce retroviruses, a retroviral vector was transfected into a packaging cell line and the transfected cells were treated with vincristine, which is an anti-cancer drug and a substrate for the MDRI gene product. This study revealed that two genes were incorporated into chromosomes of selected cells and expressed in the same cells. The production of the retrovirus was confirmed by the reverse transcription (RT)-PCR of the viral RNA. The retrovirus that was produced infected mouse fibroblast cells as well as the human U937. This study showed that packaging cells produced the retroviruses, which can infect the target cells. Once the conditions for the high infectivity of retrovirus into human cells are optimized, thus virus will be used to infect hematopoietic stem cells to co-express MDRl and HLA-B7 genes, and develop the lymphocytes that can be used for the immnogene therapy.

• BMB Reports
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• 제53권10호
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• pp.521-526
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• 2020

Endogenous retroviruses (ERVs) are retrotransposons present in various metazoan genomes and have been implicated in metazoan evolution as well as in nematodes and humans. The long terminal repeat (LTR) retrotransposons contain several regulatory sequences including promoters and enhancers that regulate endogenous gene expression and thereby control organismal development and response to environmental change. ERVs including the LTR retrotransposons constitute 8% of the human genome and less than 0.6% of the Caenorhabditis elegans (C. elegans) genome, a nematode genetic model system. To investigate the evolutionarily conserved mechanism behind the transcriptional activity of retrotransposons, we generated a transgenic worm model driving green fluorescent protein (GFP) expression using Human endogenous retroviruses (HERV)-K LTR as a promoter. The promoter activity of HERV-K LTR was robust and fluorescence was observed in various tissues throughout the developmental process. Interestingly, persistent GFP expression was specifically detected in the adult vulva muscle. Using deletion constructs, we found that the region from positions 675 to 868 containing the TATA box was necessary for promoter activity driving gene expression in the vulva. Interestingly, we found that the promoter activity of the LTR was dependent on che-1 transcription factor, a sensory neuron driver, and lin-15b, a negative regulator of RNAi and germline gene expression. These results suggest evolutionary conservation of the LTR retrotransposon activity in transcriptional regulation as well as the possibility of che-1 function in non-neuronal tissues.

• Journal of Microbiology
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• 제39권4호
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• pp.300-304
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• 2001

Epstein-Barr Virus(EBV)-transformed lymphoblastoid B cell lines, BLCL which expresse antigens, are potential antigen-presenting cells(APCs) for the induction of CTL in vitro. However transfection of BLCLs with subsequent selection by antibiotics is notoriously difficult because plating efficiencies of BLCLsare reported to be 1% or less. To generated stable transfectants of BLCLs we produced high titers of retroviruess encoding pp 65 antigen of human cytomegalovirus of foreign antigens and trans-duced them of BLCLs. The pp 65 gene was cloned into the retroviral vector pLXSN. The recombinant retroviral vector was transfected to ecotropic packaging cell line, CP&E86, and this polyclonal recom-binant retrovirus was transduced to PA317 that is amphotropic pakaging cell line. The titers of colned PA317 amphotropic retroviruses ranged from 5 to $\times$10$^{6}$ colony forming units (CFU)per ml (CFU/ml) We performed three rounds of consecutive transductions to BLCLs in order to improve the clon-ing effieiencies. The expression of recombinant HCMV-pp65 antigen was more than 20% after the final transduction. THe third-transduced BLCLs were easily selected in optimal concentration of G418. BLCLs expressing foreign antigens could be used as target cells for CTL assay and/or as APCs for induction of in vitro CTL responses specific for viral and tumor antigens.

• KSBB Journal
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• 제14권4호
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• pp.396-402
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• 1999

유전자 전달 수단으로 사용되는 레트로바이러스의 감염 효율을 증대시키는 고분자양이온의 역할을 알아보기 위해 R18 형광 물질을 이용해 고분자 양이온 중 하나인 polybrene의 유무에 따른 레트로바이러스와 세포간의 binding affinity를 직접적으로 측정하였으며 그 외의 여러 고분자물질의 레트로바이러스의 감염에 어떠한 영향을 미치는지 알아보았다. 그 결과 고분자의 전하는 레트로바이러스와 세포간의 binding affinity에는 영향을 미치지 않았으나 감염효율을 증대시키는 것은 고분자양이온 뿐이었다. 이는 고분자양이온이 레트로바이러스의 감염시 binding과정이 아닌 그 이후의 과정, 특히 internalization 과정에 영향을 미치는 것으를 나타난다. FITC가 부착된 poly-L-lysine이 세포안으로 들어가는 사실을 통해 고분자양이온의 세포안으로의 유입이 바이러스의 internalization과정에 중대한 영향을 미치는 것을 알 수 있었다. 또한 분자량이 다른 poly-L-lysine을 이용해 고분자양이온의 경우 그 분자량에 따라 최적농도가 다름을 알 수 있었다.

• 한국해양생명과학회지
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• 제1권2호
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• pp.88-94
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• 2016

Determining the infection history of living organisms is essential for understanding the evolution of infection agents with their host, particularly for key aspects such as immunity. Viruses, which can spread between individuals and often cause disease, have been widely examined. The increasing availability of fish genome sequences has provided specific insights into the diversity and host distribution of retroviruses in fish. The shortspine spurdog (Squalus mitsukurii) is an important elasmobranch species; this medium-sized dogfish typically lives at depths of 100~500 m. However, the retroviral envelope polyprotein in dogfish has not been examined. Thus, the aim of the present study was to identify and analyze the retroviral envelope polyprotein in various tissues of dogfish. The 1334-base pair full-length novel cDNA of dogfish envelope polyprotein (dEnv) was obtained by 3' and 5'-rapid amplification of cDNA end analysis from S. mitsukurii. The open reading frame showed a complete coding sequence of 815 base pairs with a deduced peptide sequence of 183 amino acids that exhibited 34~50% identity with other fish and bird species. It was also expressed according to reverse transcription and real-time polymerase chain reaction in the kidney, liver, intestine, and lung, but not in the gill. This distribution can be assessed by identifying and analyzing endogenous retroviruses in fish, which consists of three main genes: gag, pol and env. Dogfish envelope polyprotein sequence is likely important in evolution and induces rearrangements, altering the regulatory and coding sequences. This is the first report of the identification and molecular characterization of retroviral envelope polyprotein in various tissues of S. mitsukurii.

• KSBB Journal
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• 제14권5호
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• pp.593-599
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• 1999

본 연구는 제대혈과 골수로부터 얻은 조혈모세포를 재조합 retrovirus로 감염시킬 때의 최적조건을 human growth hormone (hGH)과 $\beta$-galactosidase를 발현하는 두 가지의 다른 retroviral vector를 이용하여 찾았다. Retrovirus는 자라는 세포에만 감염하는 것으로 알려져 있어 이에 대한 최적조건을 구하기 위해 세포 배양을 통해 조혈모세포의 성장곡선을 얻었으며, 또한 감염된 세포를 환자에게 다시 넣는 유전자요법에서는 이 세포가 체내에서 가능하면 조혈모세포의 기능을 가지는 것이 요구되어 이 때 얻어진 세포의 분열능을 나타내는 집락형성 세포분율을 구하였다. 우선, 세포성장에 대해 조사한 결과 초기에 넣은 세포농도가 5$\times$$10^4$세포/mL일 때 세포성장속도가 가장 빠른 것으로 나타났다. 그러나, 배양시간이 지남에 따라 집락을 형성할 수 있는 능력은 급격하게 감소하여 유전자요법을 위한 최적조건을 구하기 위해서는 이를 고려한 최적화가 필요하였다. 이를 위한 예비실험으로 감염이 잘 된다고 알려진 NIH3T3 세포에 retrovirus 상층액으로 감염시킨 결과 성공적으로 유전자가 전달된 것을 배지에 분비되는 hGH을 측정하여 확인하였다. 이러한 결과로부터 hGH을 발현하는 재조합 retrovirus는 정상적으로 작동하는 것을 확인하였다. 그러나, 조혈모세포와 retrovirus를 분비하는 packaging cell을 동시 배양하는 방법을 채택하였다. 제대혈로부터 얻은 조혈모세포와 대장균 lacZ 유전자로부터 $\beta$-galactosidase를 분비하는 packaging cell을 이용한 경우 동시배양의 경우 조혈모세포를 3일 동안 세포배양을 한 후 이 증식된 세포를 48시간 동안 동시배양하면서 감염시켰을 때 최대의 감염율을 나타내었다. 한편, 골수로부터 얻은 조혈모세포와 hGH을 분비하는 packaging cell과 동시배양시켰을 때 세포농도가 다름에도 불구하고 제대혈에서와 마찬가지로 조혈모세포를 3일 동안 세포배양한 후 48시간 동안 동시배양하는 경우에 hGH이 최대로 분비되었다. 이러한 결과로부터 세포의 source나 세포농도와 관계없이 유전자전달을 통한 단백질의 발현에 있어서 최적조건이 존재하였다. 그러나, 이러한 경우에 유전자전달이 완료되는 시점이 배양을 시작한지 5일이 되므로 집락을 형성할 수 있는 세포의 분율이 약 1/3로 감소하였다. 따라서, 이러한 결과를 유전자요법에 적용하는 경우에는 그 목적에 따라 적절한 실험조건을 선정하는 것이 필요하리라 사료된다.

• BMB Reports
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• 제36권3호
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• pp.305-311
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• 2003

A modified actinomycin D was prepared with a hydroxyl group that replaced the amino group at the chromophore 2-position, a substitution known to strongly reduce affinity for double-stranded DNA. Interactions of the modified drug on single-stranded DNAs of the defined sequence were investigated. Competition assays showed that 2-hydroxyactinomycin D has low affinity for two oligonucleotides that have high affinities ($K_a\;=\;5-10{\times}10^6\;M^{-1}$ oligomer) for 7-aminoactinomycin D and actinomycin D. Primer extension inhibition assays performed on several single-stranded DNA templates totaling around 1000 nt in length detected a single high affinity site for 2-hydroxyactinomycin D, while many high affinity binding sites of unmodified actinomycin D were found on the same templates. The sequence selectivity of 2-hydroxyactinomycin D binding is unusually high and approximates the selectivity of restriction endonucleases. Binding appears to require a complex structure, including residues well removed from the polymerase pause site.