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

BioTechniques

Sumiti Jain¹  David E. Ott¹  Lori V. Coren¹  Matthew T. Trivett¹  Claes Ohlen¹ 

[1] 1AIDS and Cancer Virus Program, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MD;

关键词: T-cell receptor;    Golden Gate cloning;    retroviral vector;    gene engineering;   

DOI  :  10.2144/000114265

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown