• Toxicological Research
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• 제17권
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• pp.157-166
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• 2001

Almost all currently available retroviral vectors based on murine leukemia virus (MLV) contain one or more viral coding sequences. Because these sequences are also present in the packaging genome, it has been suggested that homologous recombination may occur between the same nucleotide sequence in the packaging genome and the vector, resulting in the production of replication competent retrovirus (RCR). Up until now, it has been difficult to completely remove viral coding sequences since some were thought to be involved in the optimum function of the retroviral vector. For example, the gag coding sequence present in almost all available retroviral vectors has been believed to be necessary for efficient viral packaging, while the pol coding sequence present in the highly efficient vector MFG has been thought to be involved in achieving the high levels of gene expression. However, we have now developed a series of retroviral vectors that are absent of any retroviral coding sequences but produce even higher levels of gene expression without compromising viral titer. In these vectors, the intron and exon sequences from heterologous cellular or viral genes are present. When compared to the well known MLV-based vectors, some of these newly developed vectors have been shown to produce significantly higher levels of gene expression for a longer period. In an experimental system that can maximize the production of RCR, our newly constructed vectors produced an absence of RCR. These vectors should prove to be safer than other currently available retroviral vectors containing one or more viral coding sequences.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2000년도 추계 학술대회
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• pp.31-50
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• 2000

Almost all currently available retroviral vectors based on murine leukemia virus (MLV) contain one or more viral coding sequences Because these sequences are also present in the packaging genome, it has been suggested that homologous recombination may occur between the same nucleotide sequence in the packaging genome and the vector, resulting in the production of replication competent retrovirus (RCR). Up until now, it has been difficult to completely remove viral coding sequences since some were thought to be involved in the optimum function of the retroviral vector. For example, the gag coding sequence present in almost all available retroviral vectors has been believed to be necessary for efficient viral packaging, while the pol coding sequence present in the highly efficient vector MFG has been thought to be involved in achieving the high levels of gene e(pression. However, we have now developed a series of reroviral vectors that are absent of any retroviral coding sequences but produce even higher levels of gene expression without compromising viral titer. In these vectors the intron and exon sequences from heterologous cellular or viral genes are present, When compared to the well blown MLV-based vectors, some of these newly developed vectors have been shown to produce significantly higher levels of gene expression for a longer period. In an experimental system that can maximize the production of RCR, our newly constructed vectors produced an absence of RCR. These vectors should prove to be safer than other currently available retroviral vectors containing one or more viral coding sequences

• Molecules and Cells
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• 제40권5호
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• pp.339-345
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• 2017

Retroviral and lentiviral vectors are mostly pseudotyped and often purified and concentrated via ultracentrifugation. In this study, we quantified and compared the stabilities of retroviral [murine leukemia virus (MLV)-based] and lentiviral [human immunodeficiency virus (HIV)-1-based] vectors pseudotyped with relatively mechanically stable envelope proteins, vesicular stomatitis virus glycoproteins (VSVGs), and the influenza virus WSN strain envelope proteins against ultracentrifugation. Lentiviral genomic and functional particles were more stable than the corresponding retroviral particles against ultracentrifugation when pseudotyped with VSVGs. However, both retroviral and lentiviral particles were unstable when pseudotyped with the influenza virus WSN strain envelope proteins. Therefore, the stabilities of pseudotyped retroviral and lentiviral vectors against ultracentrifugation process are a function of not only the type of envelope proteins, but also the type of viral internal core (MLV or HIV-1 core). In addition, the fraction of functional viral particles among genomic viral particles greatly varied at times during packaging, depending on the type of envelope proteins used for pseudotyping and the viral internal core.

• 대한바이러스학회지
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• 제29권1호
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• pp.33-38
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• 1999

Retroviral vector provide a highly efficient method for gene transfer into eukaryotic cells. This vector system can be divided into two components; the retroviral vector itself and the retroviral packaging cell line. The key improvement in the design of these two components are, focused on two aspects; the reduction of helper virus production and high titer-virus. We used PA317 for retrovirus packaging cell line, for its high producibility of viral titer. To test the ability of the vectors to generate high titer-virus, we have chosen four different retroviral vectors; LN, LNSX, LNCX and LXSN. To test easily the viral titer, we have made recombinant construction with CD4 and CD8, checked their viral titer and stained their surface expression. LXSN which contain SV40 early promoter in front of neo gene showed best results in viral transient transfection assay, dot blot assay and surface expression. In addition, recombinant containing CD8 generally showed much higher viral titration and surface expression than CD4.

• BMB Reports
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• 제45권4호
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• pp.207-212
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• 2012

Retroviruses have often been used for gene therapy because of their capacity for the long-term expression of transgenes via stable integration into the host genome. However, retroviral integration can also result in the transformation of normal cells into cancer cells, as demonstrated by the incidence of leukemia in a recent retroviral gene therapy trial in Europe. This unfortunate outcome has led to the rapid initiation of studies examining various biological and pathological aspects of retroviral integration. This review summarizes recent findings from these studies, including the global integration patterns of various types of retroviruses, viral and cellular determinants of integration, implications of integration for gene therapy and retrovirus-mediated infectious diseases, and strategies to shift integration to safe host genomic loci. A more comprehensive and mechanistic understanding of retroviral integration processes will eventually make it possible to generate safer retroviral vector platforms in the near future.

• BMB Reports
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• 제30권4호
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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.

• 한국가축번식학회지
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• 제25권4호
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• pp.399-407
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• 2001

MII단계 난자의 위란강에 retroviral vector를 주입하여, 형질전환 난자의 생산하려는 연구가 수행되고 있다. 그러나 이러한 난자의 위란강의 크기는 매우 다양하므로, 외래유전자를 위란강에 미세주입을 할 때, 난자의 세포질에 손상을 줄 수가 있다. 이에 본 연구에서는 외래유전자 주입시 발생할 수 있는 난자세포의 손상을 최소화하기 위하여 sucrose처리법을 채택하였다. 즉 난자를 0.5%의 sucrose가 첨가된 배양액으로 처리함으로써 일정한 형태의 세포질을 유지하지 못하는 난자와 일정한 형태의 세포질을 유지하는 난자로 분류할 수 있었으며, 후자의 경우 세포질의 큰 손상 없이 retroviral vector를 난자의 위란강내에 주입할 수 있었다. 그러나 sucrose처리에 의해 선별된 난자의 수정율과 대조군의 그것 사이에는 유의차가 없었다. 또 sucrose처리에 의해 선발된 난자에 있어서 retroviral vector (LN$\beta$-EGFP and LNC-hGH) 주입 후의 분할율과 배반포발달율 같은 양상을 보였다. LN$\beta$-EGFP이 주입된 경우, 분할율과 배반포율이 81 및 25% 보였으며, LNC-hGH이 주입된 경우, 83 및 30%를 보였다. 그 결과 미세주입된 난자는 대조군과 유의적인 차이 없이 발달할 수 있었다. 게다가, hGH-gene의 결합율이 PCR 분석에 의하여 분할된 난자에서 52%를 보였으며, 또한 EGFP-gene의 발현율이 현광현미경을 총해 배반포난자 에서 34%가 관찰되었다. 이상의 결과를 종합할 매, 0.5%의 sucrose처리는 우량난자의 선발을 가능하게 하였으며, 주입유전자의 발현율이 낮아지지 않았을 뿐만 아니라, 외래유전자의 위란강내 주입시 난자에 대한 물리적 손상을 줄일 수 있으므로 발달율을 개선할 수 있는 것으로 사료된다.

• BMB Reports
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• 제55권12호
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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.

• 한국동물학회:학술대회논문집
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• 한국동물학회 2001년도 공동 춘계학술대회
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• pp.94.1-94
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• 2001

No Abstract, See Full Text

• 대한생식의학회:학술대회논문집
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• 대한불임학회 제1차 연수강좌
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• pp.229-234
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• 2000

Transgenic animal technology has provided new opportunities in many aspects of biotechnology and medicine during two decades. Several gene delivery systems including pronuclear injection, retroviral vectors, sperm vectors, and somatic cell cloning have been tried to generate new functional animals. In the future somatic cell cloning technology will be a major method in the transgenic animal production. Many factors enhancing overall transgenic efficiency should be overcome to facilitate the industrial applications of transgenic technology. Transgenic animal technology has settled down in some areas of the medicine, especially the mass production of pharmaceutical proteins and xenotransplantation. Thus, animal biotechnology will contribute to welfare of human being.