RAG-2-deficient blastocyst complementation: an assay of gene function in lymphocyte development.
We describe a system to evaluate the function of lymphocyte-specific and generally expressed genes in the differentiation and/or function of lymphocytes. RAG-2 (recombination-activating gene 2)-deficient mice have no mature B and T lymphocytes due to the inability to initiate VDJ recombination. Blastocysts from RAG-2-deficient mice generate animals with no mature B and T cells following implantation into foster mothers. However, injection of normal ES cells into RAG-2-deficient blastocysts leads to the generation of somatic chimeras with mature B and T cells all of which derive from the injected ES cells (referred to as RAG-2-deficient blastocyst complementation). Complementation of RAG-2-deficient blastocysts with mutant ES cells heterozygous for a targeted mutation that deletes all immunoglobulin heavy-chain joining (JH) gene segments (JH+/-) also leads to generation of chimeras with normal B and T cells. However, complementation with ES cells homozygous for the JH mutation (JH-/-) generates animals with normal T cells but no B cells, due to a block in B-cell development at a very early stage. Transfection of a functionally assembled mu heavy-chain gene into the JH-/- ES cells prior to blastocyst injection rescues the JH-/- mutation and allows the generation of both mature T and mature B cells. The rescued B cells express IgM but not IgD and respond normally to bacterial lipopolysaccharide stimulation by proliferating and by secreting IgM.
Chen
J
J
Howard Hughes Medical Institute, Children's Hospital, Department of Genetics, Harvard University Medical School, Boston.
Lansford
R
R
Stewart
V
V
Young
F
F
Alt
F W
FW
D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology* / Q000276:immunology; D001755:Blastocyst; D002454:Cell Differentiation; D004268:DNA-Binding Proteins; D005260:Female; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D005803:Genes, Immunoglobulin; D005816:Genetic Complementation Test; D007148:Immunoglobulin mu-Chains / Q000235:genetics*; D051379:Mice; D008817:Mice, Mutant Strains; D011506:Proteins / Q000502:physiology*; D015183:Restriction Mapping; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology
D004268:DNA-Binding Proteins; D007148:Immunoglobulin mu-Chains; D011506:Proteins; C499308:Rag2 protein, mouse; C064655:V(D)J recombination activating protein 2