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.
Howard Hughes Medical Institute, Children's Hospital, Department of Genetics, Harvard University Medical School, Boston.
J
J
Chen
R
R
Lansford
V
V
Stewart
F
F
Young
F W
FW
Alt
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