TY - JOUR
T1 - Protocols for high efficiency, stage specific retroviral transduction of murine fetal thymocytes and thymic epithelial cells
AU - Travers, Helen
AU - Anderson, Graham
AU - Gentle, Dean
AU - Jenkinson, Eric
AU - Girdlestone, John
PY - 2001/7/1
Y1 - 2001/7/1
N2 - Viral vectors have the potential to provide a fast and economic alternative to transgenic methods for manipulating gene expression in studies of immune system development and function. Although protocols exist for the infection of hematopoietic precursors and peripheral T cells in vitro, critical stages of T cell differentiation are strictly dependent upon a three-dimensional thymic architecture and their analysis poses unique technical challenges. Whole fetal thymic lobes have been used as targets for retroviral and adenoviral infection, both in situ and in vitro, but this approach does not allow for discrimination between lymphoid and stromal components. Isolated thymocytes have been infected by co-culture with viral producer cells, but under these conditions they rapidly lose their developmental potential. To overcome these problems we have combined a number of efficient techniques for retroviral production, concentration, and infection that allow us to rapidly achieve significant transduction rates of purified populations of double-negative (DN) and double-positive (DP) thymocytes, single-positive (SP) T lymphocytes, as well as fetal thymic MHC II(+) epithelial cells without the need for co-culture with viral producer cells. Reaggregate thymic organ culture (RTOC) techniques were used to assess the development and function of transduced cells in defined cellular environments. As a demonstration of the utility of these methods, CD80 (B7.1) was transduced into thymic epithelial cells and shown to allow them to mediate negative selection of DP thymocytes, and to act as antigen-presenting cells (APC) to mature T cells. The ability to genetically manipulate primary cells of a specified type and differentiation stage provides a powerful complement to RTOC techniques for the study of T cell development.
AB - Viral vectors have the potential to provide a fast and economic alternative to transgenic methods for manipulating gene expression in studies of immune system development and function. Although protocols exist for the infection of hematopoietic precursors and peripheral T cells in vitro, critical stages of T cell differentiation are strictly dependent upon a three-dimensional thymic architecture and their analysis poses unique technical challenges. Whole fetal thymic lobes have been used as targets for retroviral and adenoviral infection, both in situ and in vitro, but this approach does not allow for discrimination between lymphoid and stromal components. Isolated thymocytes have been infected by co-culture with viral producer cells, but under these conditions they rapidly lose their developmental potential. To overcome these problems we have combined a number of efficient techniques for retroviral production, concentration, and infection that allow us to rapidly achieve significant transduction rates of purified populations of double-negative (DN) and double-positive (DP) thymocytes, single-positive (SP) T lymphocytes, as well as fetal thymic MHC II(+) epithelial cells without the need for co-culture with viral producer cells. Reaggregate thymic organ culture (RTOC) techniques were used to assess the development and function of transduced cells in defined cellular environments. As a demonstration of the utility of these methods, CD80 (B7.1) was transduced into thymic epithelial cells and shown to allow them to mediate negative selection of DP thymocytes, and to act as antigen-presenting cells (APC) to mature T cells. The ability to genetically manipulate primary cells of a specified type and differentiation stage provides a powerful complement to RTOC techniques for the study of T cell development.
KW - GFP
KW - retrovirus
KW - CD80
KW - organ culture
KW - thymus
UR - http://www.scopus.com/inward/record.url?scp=0035400273&partnerID=8YFLogxK
U2 - 10.1016/S0022-1759(01)00391-X
DO - 10.1016/S0022-1759(01)00391-X
M3 - Article
C2 - 11384682
VL - 253
SP - 209
EP - 222
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1-2
ER -