南方医科大学学报 ›› 2014, Vol. 34 ›› Issue (12): 1768-.

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神经干细胞特异性PPARγ基因敲除小鼠模型的制备与鉴定

吴巧琪,章红妍,王震,林利芳,陈璐,王雪敏   

  • 出版日期:2014-12-20 发布日期:2014-12-20

Neural stem cell-specific peroxisome proliferator-activated receptor γ knockout mice:
breeding and genetic identification

  • Online:2014-12-20 Published:2014-12-20

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 breed neual stem cell-specific peroxisome proliferator-activated receptor γ (PPARγ) knockout mice.
Methods Two transgenic mouse models, namely B6.PPARγloxp/loxp and B6.Nestin-Cre were interbred, and the firstgeneration
offsprings were backcrossed with B6.PPARγloxp/loxp to obtain the second-generation mice. Genomic DNA was
extracted from the second-generation mice for PCR to amplify the loxp and Cre gene fragments followed by agarose gel
electrophoresis to verify their sizes. The mice with the PPARγloxp/loxp.Nestin-Cre (KO) genotype were selected as the neural
stem cell-specific knockout PPARγ mice, with B6.PPARγloxp/loxp (loxp) mice as the control. Tissue samples were collected
from specific regions of the mouse brain and peripheral tissue for detecting the expression of PPARγ mRNA using RT-PCR
and real-time quantitative PCR. Results and Conclusion Genotyping results showed PPARγloxp and Cre bands in the
knockout mice, which showed obviously decreased mRNA expression of PPARγ, suggesting successful establishment of
neural stem cell-specific PPARγ knockout mice. The two transgenic mice we used were fertile, and their breeding pattern
followed the laws of Mendelian inheritance.