Analysis of the expression and function of T cell differentiation antigen-CD7

1. mCD 7基因敲除小鼠的产生：使用先前报道的小鼠CD 7（mCD 7）cDNA（Yoshikawa，1993）从BALB/c小鼠脾基因组文库中分离出4个阳性克隆。这些克隆覆盖24.4 kb，含有4个外显子，插入编码小鼠CD 7抗原的内含子。通过将新霉素抗性基因插入到第一外显子中并将单纯疱疹病毒-胸苷激酶基因插入到第一外显子的5'上部区域中来产生用于产生mCD 7缺陷型小鼠的构建体。然而，mCD 7缺陷型小鼠的组织学研究显示与野生型小鼠没有明显差异。与野生型小鼠相比，mCD 7缺陷小鼠淋巴细胞的增殖试验显示出对几种有丝分裂原的相似反应性。这些结果表明，在mCD 7缺陷型小鼠中，虽然可检测到mCD 7 mRNA，但并不产生mCD 7蛋白，或者可能有另一种分子替代了mCD 7抗原的功能。2.抗小鼠CD 7抗体的制备：用重组抗原（胞浆区39个氨基酸）免疫家兔。用ELISA法检测到一种抗体与免疫后的菌体产物发生反应，在转染mCD 7 cDNA的Cos 7细胞中可检测到一条分子量为27 kDa的条带，但该抗体与可能表达mCD 7抗原的小鼠胸腺细胞不发生反应。

1.Production of mCD7 gene knock out mouse : Four positive clones from a BALB/c mouse spleen genomic library were isolated using mouse CD7 (mCD7) cDNA previously reported (Yoshikawa, 1993). These clones covered 24.4 kb containing four exons with intervening introns coding for the mouse CD7 antigen. Construct for production of mCD7 deficient mouse was produced by insertions of Neomycin resistant gene into the first exon and Herpes simplex virus-thymidin kinase gene into 5' upper region of the first exon. Histological study of mCD7 deficient mouse, however, showed no clear differences from wild type mouse. Proliferation assays of lymphocytes from mCD7 deficient mouse showed a similar reactivity to several mitogens, when compared with those from wild type mouse. These results suggest that mCD7 protein is not produced although mCD7 mRNA is detectable, or that another molecule may replace the function of mCD7 antigen in mCD7 deficient mouse.2.Production of anti-mouse CD7 antibody : Recombinant antigens (39 amino acids in the cytoplasmic domain) were used for immunization of rabbits. An antibody was found to react to the immunized bacterial product by ELISA and to detect a band with a molecular mass of 27kDa in Cos7 cells transfected with mCD7 cDNA.However, this antibody did not react to mouse thymocytes which are most likely expressing mCD7 antigen.