神经干细胞特异性PPARγ基因敲除小鼠模型的制备与鉴定

发布时间：2019-03-24 16:14

【摘要】：目的制备与鉴定神经干细胞特异性PPARγ基因敲除小鼠模型。方法将引进的2种转基因小鼠B6.PPARγloxp/loxp、B6.Nestin-Cre进行饲养并杂交繁殖,将子一代小鼠与B6.PPARγloxp/loxp小鼠回交获得子二代小鼠,提取子二代小鼠的基因组DNA,利用PCR方法扩增Cre和loxp基因片段,并进行琼脂糖凝胶电泳检测。选取基因型为B6.PPARγloxp/loxp.Nestin-Cre(KO)的小鼠即为神经干细胞特异性敲除PPARγ的敲除小鼠,另选基因型为B6.PPARγloxp/loxp(loxp)作为对照组小鼠。应用RT-PCR、实时荧光定量PCR方法鉴定神经干细胞特异性敲除PPARγ的敲除小鼠。结果敲除小鼠在基因鉴定时可以扩增得到PPARγloxp和Cre两个条带,在mRNA表型检测时脑内PPARγ表达显著低于对照组小鼠。成功获得神经干细胞敲除PPARγ基因的敲除小鼠。所购2种转基因小鼠均有繁殖能力,其繁殖符合孟德尔遗传规律。结论基于loxp-Cre系统成功构建神经干细胞特异性敲除PPARγ的基因敲除小鼠,为进一步的神经系统疾病的治疗及其机制研究提供模型基础。
[Abstract]:Objective to prepare and identify neural stem cell specific PPAR 纬 gene knockout mouse model. Methods two kinds of transgenic mice B6.PPAR 纬 loxp/loxp,B6.Nestin-Cre were bred and hybridized. The second generation mice were obtained by backcross between the first generation mice and the B6.PPAR 纬 loxp/loxp mice. The genomic DNA, of the second generation mice was extracted from the second generation mice. Cre and loxp gene fragments were amplified by PCR and detected by agarose gel electrophoresis. Mice with B6.PPAR 纬 loxp/loxp.Nestin-Cre (KO) genotype were selected as neural stem cell specific knockout mice with PPAR 纬 knockout, and B6.PPAR纬 loxP / loxp (loxp) as control mice. RT-PCR, real-time fluorescence quantitative PCR was used to identify neural stem cell specific knockout PPAR 纬 knockout mice. Results two bands of PPAR 纬 loxP and Cre were amplified in knockout mice during gene identification, and the expression of PPAR 纬 in brain was significantly lower than that in control mice when detected by mRNA phenotype. The knockout mice with PPAR 纬 gene knockout from neural stem cells were successfully obtained. The two transgenic mice had reproductive ability, and their reproduction was in accordance with Mendelian heredity law. Conclusion Gene knockout mice with neural stem cell specific knockout of PPAR 纬 are successfully constructed based on loxp-Cre system, which provides a model basis for further research on the treatment and mechanism of nervous system diseases.
【作者单位】：南方医科大学基础医学院神经生物学教研室;
【基金】：国家自然科学基金(81071016)~~
【分类号】：R-332

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