3 - RAG-2-Deficient Blastocyst Complementation

Abstract

The analysis of gene function in normal development has been facilitated by the development of methods of generating germline mutations in mice by the introduction of gene-targeted mutations into embryonic stem (ES) cells. This approach has been rewarding in studies of lymphocyte differentiation and function, because the specific immune system is dispensable if mice are kept in a pathogen-free environment. However, this approach is limited in a number of practical aspects that include expense, time, and inability to evaluate roles of many generally expressed genes. To overcome these limitations, the germline mutational approach has been modified to develop an efficient, reliable, and physiological assay for gene function in lymphocyte development that is referred to as “RAG-2-deficient blastocyst complementation.” Recombination activating gene 2 (RAG-2)-deficient blastocyst complementation is based on the finding that mice homozygous for a mutation that inactivates the RAG-2 do not produce any mature T or B lymphocytes. Lymphocyte differentiation in RAG-2-deficient mice is blocked at an early stage. This mutation does not affect the development or function of any other cell type; RAG-2-deficient mice are healthy and breed well when kept in a pathogen-free environment, allowing the requisite maintenance of large breeding colonies of these animals.