Development of Multiple Cloning Site cis-Vectors for Recombinant Adeno-Associated Virus Production
Recombinant adeno-associated virus (rAAV) has become a very popular gene therapy vector in the past several years. A cis-plasmid is used to generate the rAAV stocks. In this plasmid, the entire expression cassette is incorporated between two AAV inverted terminal repeats. The construction of cis-pla...
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| Format: | Article |
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Taylor & Francis Group
2002-09-01
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| Series: | BioTechniques |
| Online Access: | https://www.future-science.com/doi/10.2144/02333dd03 |
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| _version_ | 1850152559204892672 |
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| author | Yongping Yue Dongsheng Duan |
| author_facet | Yongping Yue Dongsheng Duan |
| author_sort | Yongping Yue |
| collection | DOAJ |
| description | Recombinant adeno-associated virus (rAAV) has become a very popular gene therapy vector in the past several years. A cis-plasmid is used to generate the rAAV stocks. In this plasmid, the entire expression cassette is incorporated between two AAV inverted terminal repeats. The construction of cis-plasmid has been problematic because of the high-frequency recombination of the viral inverted terminal repeats. Here we describe the design and construction of several multiple cloning site cis-plasmids that are driven by five different promoters, including the ubiquitous cytomegalovirus enhancer/chicken β-actin (CAG), cytomegalovirus (CMV), rous sarcoma virus (RSV), simian virus 40 (SV40), and a muscle-specific promoter (CK6). The application of these multiple cloning site cis-plasmids improves the cloning efficiency. As an example of the utilization of these multiple cloning site vectors, the prokaryotic β-galactosidase cDNA was cloned in the multiple cloning site cis-plasmids. High-level rAAV-mediated β-galactosidase expression was achieved in HeLa cells from CAG, CMV, RSV, and SV40 promoters, respectively, but not from the CK6 promoter. In vivo application in the adult mdx mouse (mouse model for Duchenne muscular dystrophy) muscle revealed efficient transgene expression from CMV and CK6 promoters, followed by CAG and RSV promoters. The SV40 promoter was the least efficient. |
| format | Article |
| id | doaj-art-c9cf8de822ad4891b688853a73e95b92 |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2002-09-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-c9cf8de822ad4891b688853a73e95b922025-08-20T02:25:57ZengTaylor & Francis GroupBioTechniques0736-62051940-98182002-09-0133367267810.2144/02333dd03Development of Multiple Cloning Site cis-Vectors for Recombinant Adeno-Associated Virus ProductionYongping Yue0Dongsheng Duan11The University of Missouri, Columbia, MO, USA1The University of Missouri, Columbia, MO, USARecombinant adeno-associated virus (rAAV) has become a very popular gene therapy vector in the past several years. A cis-plasmid is used to generate the rAAV stocks. In this plasmid, the entire expression cassette is incorporated between two AAV inverted terminal repeats. The construction of cis-plasmid has been problematic because of the high-frequency recombination of the viral inverted terminal repeats. Here we describe the design and construction of several multiple cloning site cis-plasmids that are driven by five different promoters, including the ubiquitous cytomegalovirus enhancer/chicken β-actin (CAG), cytomegalovirus (CMV), rous sarcoma virus (RSV), simian virus 40 (SV40), and a muscle-specific promoter (CK6). The application of these multiple cloning site cis-plasmids improves the cloning efficiency. As an example of the utilization of these multiple cloning site vectors, the prokaryotic β-galactosidase cDNA was cloned in the multiple cloning site cis-plasmids. High-level rAAV-mediated β-galactosidase expression was achieved in HeLa cells from CAG, CMV, RSV, and SV40 promoters, respectively, but not from the CK6 promoter. In vivo application in the adult mdx mouse (mouse model for Duchenne muscular dystrophy) muscle revealed efficient transgene expression from CMV and CK6 promoters, followed by CAG and RSV promoters. The SV40 promoter was the least efficient.https://www.future-science.com/doi/10.2144/02333dd03 |
| spellingShingle | Yongping Yue Dongsheng Duan Development of Multiple Cloning Site cis-Vectors for Recombinant Adeno-Associated Virus Production BioTechniques |
| title | Development of Multiple Cloning Site cis-Vectors for Recombinant Adeno-Associated Virus Production |
| title_full | Development of Multiple Cloning Site cis-Vectors for Recombinant Adeno-Associated Virus Production |
| title_fullStr | Development of Multiple Cloning Site cis-Vectors for Recombinant Adeno-Associated Virus Production |
| title_full_unstemmed | Development of Multiple Cloning Site cis-Vectors for Recombinant Adeno-Associated Virus Production |
| title_short | Development of Multiple Cloning Site cis-Vectors for Recombinant Adeno-Associated Virus Production |
| title_sort | development of multiple cloning site cis vectors for recombinant adeno associated virus production |
| url | https://www.future-science.com/doi/10.2144/02333dd03 |
| work_keys_str_mv | AT yongpingyue developmentofmultiplecloningsitecisvectorsforrecombinantadenoassociatedvirusproduction AT dongshengduan developmentofmultiplecloningsitecisvectorsforrecombinantadenoassociatedvirusproduction |