论文部分内容阅读
目的通过制备Rtn4-A/B基因敲除小鼠,探索Rtn4-B基因的生物学功能。方法用细菌人工染色体(BAC)载体构建Rtn4-A/B基因打靶载体并使其线性化,通过电转化法将其转入129SvEv品系雄性小鼠胚胎干细胞(embryonic stem cell,ES细胞)。将正确同源重组的ES细胞注射入C57BL/6J小鼠囊胚腔,繁育出嵌合体小鼠后,进一步繁殖以获得杂合子小鼠。抽提小鼠尾尖组织DNA,采用PCR法鉴定小鼠的基因型。结果基因打靶后,得到14个发生双臂正确同源重组ES细胞克隆。利用阳性ES细胞克隆进行囊胚内显微注射,得到5只嵌合率大于50%的雄鼠,最终繁育得到4只Rtn4-A+/-B+/-杂合子小鼠。结论利用ES细胞基因打靶、同源重组等方法,成功获得Rtn4-A/B基因敲除杂合子小鼠。
Objective To explore the biological function of Rtn4-B gene by preparing Rtn4-A / B knockout mice. Methods The gene targeting vector of Rtn4-A / B was constructed by bacterial artificial chromosome (BAC) vector and linearized. The vector was transformed into 129SvEv strain embryonic stem cells (ES cells) by electroporation. ES cells of the correct homologous recombination were injected into the blastocoele of C57BL / 6J mice to produce chimeric mice, which were further propagated to obtain heterozygous mice. The DNA of mouse tail tissue was extracted and the genotype of mouse was identified by PCR. Results After gene targeting, 14 clones of ES cell clones with the correct double arms were obtained. Microinjection into the blastocysts using positive ES cell clones resulted in 5 male mice with a chimeric rate of greater than 50%, and eventually 4 Rtn4-A +/- B +/- heterozygous mice were bred. Conclusions The Rtn4-A / B knockout heterozygous mice were successfully obtained by ES cell gene targeting and homologous recombination.