Analysis of Gene Function in Lymphocytes by RAG-2-Deficient Blastocyst Complementation

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Publisher Summary The chapter discusses the recombination activating gene-2 (RAG-2)-deficient blastocyst complementation system that was developed to assay gene function in lymphocytes providing an efficient complementary assay to the germline mutational approach. RAG-2-deficient blastocyst complementation has been used to assay the role of a diverse group of genes and loci at all aspects of lymphocyte development. The chapter provides an efficient complementary approach to the germline mutation method. Because of its easy complementation, the approach is expected to be utilized more widely for studying structure function relationships and elucidating signal transduction pathways in the developing animals.RAG-2 is specifically expressed in precursor lymphocytes for the assembly of T cell receptor (TCR) and immunoglobulin (Ig) variable region genes. Experimentally, both the germline mutational approach and the RAG- 2-deficient blastocyst complementation require the generation of heterozygous mutant embryonic stein (ES) cells by homologous recombination and the generation of chimeric mice. However, they differ in generating homozygous mutation for analysis. Using the germline mutational approach, homozygous mutation is achieved through breeding of chimeric mice for germline transmission of the mutant allele and then of heterozygous mutant mice. In contrast, by RAG-2-deficient blastocyst complementation, homozygous mutation is obtained by manipulating heterozygous mutant ES cells in vitro. Because of this difference, RAG-2-deficient blastocyst complementation has some unique advantages.