Zhou Jiang, Cheng Xuan, Sun Yanxun, Huang Peitang, Huang Cuifen, Yang Xiao
Genetic Laboratory of Development and Diseases, Institute of Biotechnology, 100071, Beijing, China.
Sci China C Life Sci. 2002 Apr;45(2):129-37. doi: 10.1360/02yc9015.
Smads is a new gene family in transforming growth factor-beta (TGF- beta) signaling pathway. Smad2 mutated in multiple human tumors and may be a candidate tumor suppressor gene. Targeted disruption of murine Smad2 gene resulted in embryonic lethality at E6.5. To study the function of Smad2 in vertebrate organgenesis and tumorigenesis, we constructed the Smad2 conditional targeting vector in which two LoxP sequences were placed to flank the sequences encoding the C terminal functional domain of Smad2. The validity of the LoxP sites in the targeting construct was tested in E. coli that express the Cre recombinase constitutively. The vector was electroporated into ES cells and 3 targeted ES cell clones were obtained by Southern blot screening. Targeted ES cells were introduced into C57BL/6J blastocysts by microinjection to generate germ-line chimeras. Genotyping analysis showed that 2 progeny among these chimeras carried the Smad2 conditional targeted allele. The establishment of Smad2 conditional gene targeting mouse has laid a solid foundation for producing the tissue specific Smad2 gene knockout mice.
Smads是转化生长因子-β（TGF-β）信号通路中的一个新基因家族。Smad2在多种人类肿瘤中发生突变，可能是一种候选肿瘤抑制基因。小鼠Smad2基因的靶向破坏导致在胚胎第6.5天出现胚胎致死性。为了研究Smad2在脊椎动物器官发生和肿瘤发生中的功能，我们构建了Smad2条件性靶向载体，其中两个LoxP序列位于编码Smad2 C末端功能域的序列两侧。在组成型表达Cre重组酶的大肠杆菌中测试了靶向构建体中LoxP位点的有效性。将该载体电穿孔导入胚胎干细胞，通过Southern印迹筛选获得了3个靶向胚胎干细胞克隆。通过显微注射将靶向胚胎干细胞引入C57BL/6J囊胚中以产生种系嵌合体。基因分型分析表明，这些嵌合体中有2个子代携带Smad2条件性靶向等位基因。Smad2条件性基因靶向小鼠的建立为产生组织特异性Smad2基因敲除小鼠奠定了坚实的基础。
