RAG-2-DEFICIENT BLASTOCYST COMPLEMENTATION - AN ASSAY OF GENE-FUNCTION IN LYMPHOCYTE DEVELOPMENT

被引:329
作者
CHEN, JZ
LANSFORD, R
STEWART, V
YOUNG, F
ALT, FW
机构
[1] HARVARD UNIV,CHILDRENS HOSP,SCH MED,HOWARD HUGHES MED INST,300 LONGWOOD AVE,BOSTON,MA 02115
[2] CTR BLOOD RES,BOSTON,MA 02115
关键词
RECOMBINATION-ACTIVATING GENE; EMBRYONIC STEM CELLS; TARGETED MUTATION; SOMATIC CHIMERAS; LYMPHOCYTE DIFFERENTIATION;
D O I
10.1073/pnas.90.10.4528
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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 (J(H)) gene segments (J(H)+/-) also leads to generation of chimeras with normal B and T cells. However, complementation with ES cells homozygous for the JH mutation (J(H)-/-) 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 J(H)-/- ES cells prior to blastocyst injection rescues the J(H)-/- 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.
引用
收藏
页码:4528 / 4532
页数:5
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