Establishment of efficient reaggregation culture system for gene transfection into immature T cells by retroviral vectors

To overcome low efficiency of retroviral infection into immature T cells, w e modified reaggregation fetal thymus organ culture by closely packed co-cu lture wi th virus-producing cells (VPC). The viral vector was constructed i n chimeric vector, pMX, with IRES and tailless-rat do? as a surface marker of infected cells. A rearranged TCR beta gene (V beta 8.2) was Further inse rted into the construct for investigating effect of tlx introduced gene in T cell development. Using this system, we succeeded to transfer the viral v ector into immature thymocytes at a remarkably higher efficiency compared t o conventional methods using medium containing retrovirus. Moreover: the in troduced TCR beta gene was expressed on thymocytes of RAG2-deficient mice t o 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 tr ansfer into immature T cells. (C) 2000 Elsevier Science B.V. All rights res erved.