本文選題：XBPs　切入點(diǎn)：非折疊蛋白反應(yīng)/內(nèi)質(zhì)網(wǎng)應(yīng)激　出處：《中華腎病研究電子雜志》2016年06期

【摘要】：目的獲得可進(jìn)行條件性過表達(dá)XBP1s基因的Rosa 26定點(diǎn)敲入雜合子小鼠。方法通過In-Fusion Cloning的方法構(gòu)建胚胎干細(xì)胞(ES細(xì)胞)打靶載體,并進(jìn)行線性化,電轉(zhuǎn)染JM8A3 ES細(xì)胞。藥物篩選獲得抗性ES細(xì)胞克隆,經(jīng)長片段PCR鑒定獲得正確同源重組的陽性克隆。陽性ES細(xì)胞經(jīng)克隆擴(kuò)增后,注射入C57BL/6J小鼠的囊胚中,獲得嵌合鼠,篩選出高比例嵌合小鼠與野生型C57BL/6J小鼠交配后獲得陽性F1代小鼠,并分別進(jìn)行PCR和測序鑒定。結(jié)果經(jīng)酶切鑒定證明打靶質(zhì)粒構(gòu)建成功,經(jīng)胚胎干細(xì)胞打靶共獲得144個抗性ES細(xì)胞克隆,通過長片段PCR的方式對同源重組陽性克隆進(jìn)行篩選和克隆經(jīng)測序確認(rèn),共獲得21個正確同源重組的陽性克隆。陽性ES細(xì)胞克隆E3、C6經(jīng)擴(kuò)增后注射C57BL/6J小鼠囊胚96個,通過胚胎移植,共獲得2只高嵌合雄鼠,與野生型C57BL/6J小鼠交配后獲得3只F1代雜合小鼠,經(jīng)PCR及測序鑒定正確。結(jié)論成功建立了XBP1s基因的Rosa26定點(diǎn)敲入F1代雜合子小鼠,為未來獲得免疫細(xì)胞、腎臟固有細(xì)胞及腹膜間皮細(xì)胞XBP1s條件性敲入小鼠奠定了基礎(chǔ)。
[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.
【作者單位】： 第四軍醫(yī)大學(xué)西京醫(yī)院腎臟內(nèi)科;上海南方模式生物發(fā)展有限公司;
【基金】：國家自然科學(xué)基金(81470993;81272621) 百特公司腹膜透析專項課題(CHN-RENAL-IIS-2012-039) 西京醫(yī)院新技術(shù)新業(yè)務(wù)(XJGX15Y45)
【分類號】：R-332
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本文編號：1654644