title: 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 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.
authors.affiliation: AIDS and Cancer Virus Program, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MD.
authors.forename: Lori V
authors.initials: LV
authors.lastname: Coren
authors.affiliation: AIDS and Cancer Virus Program, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MD.
authors.forename: Sumiti
authors.initials: S
authors.lastname: Jain
authors.affiliation: AIDS and Cancer Virus Program, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MD.
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Trivett
authors.affiliation: AIDS and Cancer Virus Program, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MD.
authors.forename: Claes
authors.initials: C
authors.lastname: Ohlen
authors.affiliation: AIDS and Cancer Virus Program, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MD.
authors.forename: David E
authors.initials: DE
authors.lastname: Ott
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D008253:Macaca mulatta / Q000235:genetics; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000096:biosynthesis / Q000235:genetics*; D013601:T-Lymphocytes / Q000378:metabolism*; D014161:Transduction, Genetic
chemical_list: D016693:Receptors, Antigen, T-Cell, alpha-beta
keywords: Golden Gate cloning; T-cell receptor; gene engineering; retroviral vector