Establishment of efficient reaggregation culture system for gene transfection into immature T cells by retroviral vectors |
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Authors: | Hozumi K Ohtsuka R Suzuki D Ando K Ito M Nishimura T Merkenschlager M Habu S |
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Affiliation: | Department of Immunology, Tokai University School of Medicine, Bohseidai, Isehara, Japan. |
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Abstract: | To overcome low efficiency of retroviral infection into immature T cells, we modified reaggregation fetal thymus organ culture by closely packed co-culture with virus-producing cells (VPC). The viral vector was constructed in chimeric vector, pMX, with IRES and tailless-rat CD2 as a surface marker of infected cells. A rearranged TCR beta gene (Vbeta8.2) was further inserted into the construct for investigating effect of the introduced gene in T cell development. Using this system, we succeeded to transfer the viral vector into immature thymocytes at a remarkably higher efficiency compared to conventional methods using medium containing retrovirus. Moreover, the introduced TCR beta gene was expressed on thymocytes of RAG2-deficient mice to induce in the transition of CD4-CD8- double-negative (DN) into CD4+CD8+ double-positive (DP) cells by transducing beta-selection signaling. Thus, our modified reaggregation culture system is useful for studying the molecular mechanism of T cell development due to a highly efficient gene transfer into immature T cells. |
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