Germinal Center Formation with Retrovirally Transduced B Cells for Determining the Role of Specific Molecules In Vivo.

Abstract

Retrovirus-mediated gene transfer has become a powerful tool to investigate roles of specific molecules in B cells, due to its efficiency and expeditiousness. This technology is applicable to activated B cells in order to determine effects of a gene of interest during germinal center (GC) reactions in combination with adoptive transfer. To achieve this, B cells derived from SWHEL mice expressing hen egg lysozyme (HEL)-specific B cell receptors (BCR) are stimulated with HEL antigen in vivo and then with anti-CD40 antibody ex vivo. These cells are then transduced with a retrovirus allowing bicistronic co-expression of a gene of interest and GFP, enabling differentiation of transgene positive cells. The retrovirally transduced cells are then adoptively transferred into immunized CD45.1+ congenic recipient mice, to enable differentiation between donor and host cells. This chapter describes methods for (1) activation of HEL-specific mature B cells, (2) retroviral transduction of the activated B cells, (3) adoptive transfer of the cells into recipients, and (4) analysis of the resultant mice by flow cytometry.