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  • Title: Retroviral vectors for homologous recombination provide efficient cloning and expression in mammalian cells.
    Author: Kobayashi E, Kishi H, Ozawa T, Horii M, Hamana H, Nagai T, Muraguchi A.
    Journal: Biochem Biophys Res Commun; 2014 Feb 14; 444(3):319-24. PubMed ID: 24462869.
    Abstract:
    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.
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