Retroviral vectors for homologous recombination provide efficient cloning and expression in mammalian cells.
Homologous recombination technologies enable high-throughput cloning and the seamless insertion of any DNA fragment into expression vectors. Additionally, retroviral vectors offer a fast and efficient method for transducing and expressing genes in mammalian cells, including lymphocytes. However, homologous recombination cannot be used to insert DNA fragments into retroviral vectors; retroviral vectors contain two homologous regions, the 5'- and 3'-long terminal repeats, between which homologous recombination occurs preferentially. In this study, we have modified a retroviral vector to enable the cloning of DNA fragments through homologous recombination. To this end, we inserted a bacterial selection marker in a region adjacent to the gene insertion site. We used the modified retroviral vector and homologous recombination to clone T-cell receptors (TCRs) from single Epstein Barr virus-specific human T cells in a high-throughput and comprehensive manner and to efficiently evaluate their function by transducing the TCRs into a murine T-cell line through retroviral infection. In conclusion, the modified retroviral vectors, in combination with the homologous recombination method, are powerful tools for the high-throughput cloning of cDNAs and their efficient functional analysis.
Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
Eiji
E
Kobayashi
Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan. Electronic address: immkishi@med.u-toyama.ac.jp.
Hiroyuki
H
Kishi
Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
Tatsuhiko
T
Ozawa
Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
Masae
M
Horii
Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
Hiroshi
H
Hamana
Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
Terumi
T
Nagai
Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
Atsushi
A
Muraguchi
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005822:Genetic Vectors; D057166:High-Throughput Screening Assays; D051379:Mice; D011948:Receptors, Antigen, T-Cell / Q000235:genetics; D011995:Recombination, Genetic; D012190:Retroviridae / Q000235:genetics*; D013601:T-Lymphocytes / Q000378:metabolism
D018076:DNA, Complementary; D011948:Receptors, Antigen, T-Cell
High-throughput cloning; Homologous recombination; Retroviral vector