Production of retroviral constructs for effective transfer and expression of T-cell receptor genes using Golden Gate cloning
Here we present an improved strategy for producing T-cell receptor (TCR)-expressing retroviral vectors using a Golden Gate cloning strategy. This method takes advantage of the modular nature of TCR genes by directly amplifying TCR α and β variable regions from RNA or cDNA, then cloning and fusing th...
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2015-03-01
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| Series: | BioTechniques |
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| Online Access: | https://www.future-science.com/doi/10.2144/000114265 |
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| author | Lori V. Coren Sumiti Jain Matthew T. Trivett Claes Ohlen David E. Ott |
| author_facet | Lori V. Coren Sumiti Jain Matthew T. Trivett Claes Ohlen David E. Ott |
| author_sort | Lori V. Coren |
| collection | DOAJ |
| description | Here we present an improved strategy for producing T-cell receptor (TCR)-expressing retroviral vectors using a Golden Gate cloning strategy. This method takes advantage of the modular nature of TCR genes by directly amplifying TCR α and β variable regions from RNA or cDNA, then cloning and fusing them with their respective constant region genes resident in a retroviral TCR expression vector. Our one-step approach greatly streamlines the TCR vector production process in comparison to the traditional three-step procedure that typically involves cloning whole TCR genes, producing a TCR expression cassette, and constructing a retroviral construct. To date, we have generated TCR vectors that transferred seven functional human/rhesus macaque TCRs into primary T cells. The approach also holds promise for the assembly of other genes with defined variable regions, such as immunoglobulins. |
| format | Article |
| id | doaj-art-bc0005e9fbe34f0d842482bead6daed8 |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2015-03-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-bc0005e9fbe34f0d842482bead6daed82025-08-20T02:25:50ZengTaylor & Francis GroupBioTechniques0736-62051940-98182015-03-0158313513910.2144/000114265Production of retroviral constructs for effective transfer and expression of T-cell receptor genes using Golden Gate cloningLori V. Coren0Sumiti Jain1Matthew T. Trivett2Claes Ohlen3David E. Ott41AIDS and Cancer Virus Program, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MD1AIDS and Cancer Virus Program, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MD1AIDS and Cancer Virus Program, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MD1AIDS and Cancer Virus Program, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MD1AIDS and Cancer Virus Program, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MDHere we present an improved strategy for producing T-cell receptor (TCR)-expressing retroviral vectors using a Golden Gate cloning strategy. This method takes advantage of the modular nature of TCR genes by directly amplifying TCR α and β variable regions from RNA or cDNA, then cloning and fusing them with their respective constant region genes resident in a retroviral TCR expression vector. Our one-step approach greatly streamlines the TCR vector production process in comparison to the traditional three-step procedure that typically involves cloning whole TCR genes, producing a TCR expression cassette, and constructing a retroviral construct. To date, we have generated TCR vectors that transferred seven functional human/rhesus macaque TCRs into primary T cells. The approach also holds promise for the assembly of other genes with defined variable regions, such as immunoglobulins.https://www.future-science.com/doi/10.2144/000114265T-cell receptorGolden Gate cloningretroviral vectorgene engineering |
| spellingShingle | Lori V. Coren Sumiti Jain Matthew T. Trivett Claes Ohlen David E. Ott Production of retroviral constructs for effective transfer and expression of T-cell receptor genes using Golden Gate cloning BioTechniques T-cell receptor Golden Gate cloning retroviral vector gene engineering |
| title | Production of retroviral constructs for effective transfer and expression of T-cell receptor genes using Golden Gate cloning |
| title_full | Production of retroviral constructs for effective transfer and expression of T-cell receptor genes using Golden Gate cloning |
| title_fullStr | Production of retroviral constructs for effective transfer and expression of T-cell receptor genes using Golden Gate cloning |
| title_full_unstemmed | Production of retroviral constructs for effective transfer and expression of T-cell receptor genes using Golden Gate cloning |
| title_short | Production of retroviral constructs for effective transfer and expression of T-cell receptor genes using Golden Gate cloning |
| title_sort | production of retroviral constructs for effective transfer and expression of t cell receptor genes using golden gate cloning |
| topic | T-cell receptor Golden Gate cloning retroviral vector gene engineering |
| url | https://www.future-science.com/doi/10.2144/000114265 |
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