Protocols for high efficiency, stage-specific retroviral transduction of murine fetal thymocytes and thymic epithelial cells

Viral vectors have the potential to provide a fast and economic alternative to transgenic methods for manipulating gene expression in studies of immun e system development and function. Although protocols exist for the infecti on of hematopoietic precursors and peripheral T cells in vitro, critical st ages of T cell differentiation are strictly dependent upon a three-dimensio nal thymic architecture and their analysis poses unique technical challenge s. Whole fetal thymic lobes have been used as targets for retroviral and ad enoviral infection, both in situ and in vitro, bur this approach does not a llow for discrimination between lymphoid and stromal components. Isolated t hymocytes have been infected by co-culture with viral producer cells, but u nder these conditions they rapidly lose their developmental potential. To o vercome these problems we have combined a number of efficient techniques fo r retroviral production, concentration, and infection that allow us to rapi dly achieve significant transduction rates of purified populations of doubl e-negative (DN) and double-positive (DP) thymocytes. single-positive (SP)T lymphocytes, as well as fetal thymic MHC II+ epithelial cells without the n eed for co-culture with viral producer cells. Reaggreagate thymic organ cul ture (RTOC) techniques were used to assess the development and function of transduced cells in defined cellular environments. As a demonstration of th e utility of these methods, CD80 (B7.1) was transduced into thymic epitheli al cells and shown to allow them to mediate negative selection of DP thymoc ytes, and to act as antigen-presenting cells (APC) to mature T cells. The a bility to genetically manipulate primary cells of a specified type and diff erentiation stage provides a powerful complement to RTOC techniques for the study of T cell development. (C) 2001 Elsevier Science B.V. All rights res erved.