Production of retroviral constructs for effective transfer and expression of T-cell receptor genes using Golden Gate cloning

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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Bibliographic Details
Main Authors: Lori V. Coren, Sumiti Jain, Matthew T. Trivett, Claes Ohlen, David E. Ott
Format: Article
Language:English
Published: Taylor & Francis Group 2015-03-01
Series:BioTechniques
Subjects:
T-cell receptor
Golden Gate cloning
retroviral vector
gene engineering
Online Access:https://www.future-science.com/doi/10.2144/000114265
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Summary: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.
ISSN:0736-6205
1940-9818

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