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


ABSTRACT: 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 ? and ? 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.

SUBMITTER: Coren LV 

PROVIDER: S-EPMC4827251 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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

Coren Lori V LV   Jain Sumiti S   Trivett Matthew T MT   Ohlen Claes C   Ott David E DE  

BioTechniques 20150301 3


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 traditio  ...[more]

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