XBP1s条件性过表达定点敲入小鼠模型的建立

发布时间：2018-03-23 18:24

  本文选题：XBPs　切入点：非折叠蛋白反应/内质网应激　出处：《中华肾病研究电子杂志》2016年06期

【摘要】：目的获得可进行条件性过表达XBP1s基因的Rosa 26定点敲入杂合子小鼠。方法通过In-Fusion Cloning的方法构建胚胎干细胞(ES细胞)打靶载体,并进行线性化,电转染JM8A3 ES细胞。药物筛选获得抗性ES细胞克隆,经长片段PCR鉴定获得正确同源重组的阳性克隆。阳性ES细胞经克隆扩增后,注射入C57BL/6J小鼠的囊胚中,获得嵌合鼠,筛选出高比例嵌合小鼠与野生型C57BL/6J小鼠交配后获得阳性F1代小鼠,并分别进行PCR和测序鉴定。结果经酶切鉴定证明打靶质粒构建成功,经胚胎干细胞打靶共获得144个抗性ES细胞克隆,通过长片段PCR的方式对同源重组阳性克隆进行筛选和克隆经测序确认,共获得21个正确同源重组的阳性克隆。阳性ES细胞克隆E3、C6经扩增后注射C57BL/6J小鼠囊胚96个,通过胚胎移植,共获得2只高嵌合雄鼠,与野生型C57BL/6J小鼠交配后获得3只F1代杂合小鼠,经PCR及测序鉴定正确。结论成功建立了XBP1s基因的Rosa26定点敲入F1代杂合子小鼠,为未来获得免疫细胞、肾脏固有细胞及腹膜间皮细胞XBP1s条件性敲入小鼠奠定了基础。
[Abstract]:Objective to obtain Rosa 26 knockout heterozygous mice with conditional overexpression of XBP1s gene. Methods the target vector of embryonic stem cells (es cells) was constructed by In-Fusion Cloning and linearized. After electrotransfection of JM8A3 es cells, resistant es cell clones were obtained by drug screening, and correct homologous recombination positive clones were obtained by long fragment PCR identification. After cloning and amplification, positive es cells were injected into the blastocysts of C57BL/6J mice to obtain chimeric mice. High proportion of chimeric mice were mated with wild-type C57BL/6J mice to obtain positive F1 generation mice and identified by PCR and sequencing. Results the target plasmids were successfully constructed by restriction endonuclease digestion. A total of 144 resistant es cell clones were obtained by targeting embryonic stem cells. Homologous recombination positive clones were screened by long fragment PCR and confirmed by sequencing. A total of 21 positive clones with correct homologous recombination were obtained, and the positive es cell clones were amplified and injected into 96 C57BL/6J mouse blastocysts. Two highly chimeric male mice were obtained by embryo transfer. After mating with wild-type C57BL/6J mice, three F1 hybrid mice were obtained, which were identified correctly by PCR and sequencing. Conclusion the Rosa26 knockout of XBP1s gene into F1 generation heterozygote mice was successfully established for the purpose of obtaining immune cells in the future. The XBP1s conditioned knockout mice of renal intrinsic cells and peritoneal mesothelial cells laid the foundation.
【作者单位】： 第四军医大学西京医院肾脏内科;上海南方模式生物发展有限公司;
【基金】：国家自然科学基金(81470993;81272621) 百特公司腹膜透析专项课题(CHN-RENAL-IIS-2012-039) 西京医院新技术新业务(XJGX15Y45)
【分类号】：R-332
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本文编号：1654644