RAG-2-DEFICIENT BLASTOCYST COMPLEMENTATION - AN ASSAY OF GENE-FUNCTION IN LYMPHOCYTE DEVELOPMENT
RAG-2-DEFICIENT BLASTOCYST COMPLEMENTATION - AN ASSAY OF GENE-FUNCTION IN LYMPHOCYTE DEVELOPMENT
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DOI：
10.1073/pnas.90.10.4528
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发表时间：
1993-05-15
影响因子：
11.1
通讯作者：
ALT, FW
中科院分区：
文献类型：
--
作者：
CHEN, JZ;LANSFORD, R;ALT, FW
我们描述了一种评估淋巴细胞特异性和普遍表达基因在淋巴细胞分化和/或功能中的功能的系统。 RAG-2（重组激活基因 2）缺陷小鼠由于无法启动 VDJ 重组而没有成熟的 B 和 T 淋巴细胞。 RAG-2缺陷小鼠的囊胚在植入养母体内后产生的动物没有成熟的B细胞和T细胞。然而，将正常 ES 细胞注射到 RAG-2 缺陷型囊胚中会导致产生具有成熟 B 细胞和 T 细胞的体细胞嵌合体，所有这些嵌合体均源自注射的 ES 细胞（称为 RAG-2 缺陷型囊胚互补）。 RAG-2缺陷型囊胚与突变型ES细胞的互补，该突变型ES细胞具有靶向突变的杂合性，该突变删除了所有免疫球蛋白重链连接(J(H))基因片段(J(H)+/-)，也导致与正常B和T细胞的嵌合体的产生。然而，由于 B 细胞发育在极早期阶段受到阻碍，与 JH 突变 (J(H)-/-) 纯合 ES 细胞互补会产生具有正常 T 细胞但没有 B 细胞的动物。在囊胚注射之前将功能组装的 mu 重链基因转染至 J(H)-/- ES 细胞中可挽救 J(H)-/- 突变并允许生成成熟 T 细胞和成熟 B 细胞。获救的 B 细胞表达 IgM 但不表达 IgD，并通过增殖和分泌 IgM 对细菌脂多糖刺激做出正常反应。
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.