Use of Stem Cell Radiation Chimeras to Analyze How Domains of Specific Proteins Impact on Murine NK Cell Development In Vivo

Abstract

Although the use of mutant mice has been extremely useful in identifying those proteins and molecules specifically required for the development of NK cells, the establishment of a well-defined protocol to replicate in vitro the major steps corresponding to the process of NK cell differentiation and maturation has enabled us to dissect the molecular events governing certain aspects of NK cell development. This chapter describes a protocol that combines both the use of mutant mice and the in vitro bone marrow (BM) culture system for examining the role of proteins and their putative signaling domains in NK cell development. BM-derived Lin–c-kit⁺ stem cells expressing the protein of interest are first cultured for 6 days in a cocktail of cytokines that promote lymphoid development. The semi-differentiated cells are then transplanted into mice to complete their development in vivo. While all hematopoietic lineages can develop from these transplanted cells, we focus primarily on assessing the effect of the protein on the production of NK cells, as well as the acquisition of Ly49 receptors. The most prevalent advantage of this method is the ability to potentially link signaling regulators to known aspects of NK cell development.