首都医科大学学报 ›› 2013, Vol. 34 ›› Issue (1): 105-109.doi: 10.3969/j.issn.1006-7795.2013.01.020

• 基础研究 • 上一篇    下一篇

GAP-43高表达转基因小鼠的建立

黄蕊, 赵君朋, 文玉军, 徐群渊   

  1. 首都医科大学北京神经科学研究所 北京神经再生修复研究重点实验室 教育部神经变性病重点实验室, 北京 100069
  • 收稿日期:2012-09-06 出版日期:2013-02-21 发布日期:2013-02-25
  • 通讯作者: 徐群渊 E-mail:xuqy@ccmu.edu.cn
  • 基金资助:

    国家重点基础研究发展计划资助项目(2007CB947704)。

Establishment of transgenic mice with GAP-43 over expression

HUANG Rui, ZHAO Junpeng, WEN Yujun, XU Qunyuan   

  1. Institute for Neuroscience, Beijing Center of Neural Regeneration & Repair, Capital Medical University, Key Laboratory of Neurodegenerative Diseases, Ministry of Education, Beijing 100069, China
  • Received:2012-09-06 Online:2013-02-21 Published:2013-02-25
  • Supported by:

    This study was supported by National Key Basic Research and Development Program Funded Projects(2007CB947704).

摘要:

目的 构建GAP-43(growth associated protein-43)高表达转基因小鼠,为进一步研究GAP-43对神经发生及损伤修复的作用机制提供基础。方法构建pCEP4-PDGF-GAP-43转基因构件,通过原核显微注射方法将线性化、纯化后的外源质粒pCEP4-PDGF-GAP-43 注射入C57小鼠受精卵中,胚胎移植至同期发情的假孕受体母鼠输卵管内,获得子代小鼠。用PCR方法检测子代鼠尾基因组DNA,通过Western blotting法检测GAP-43基因表达,并通过免疫荧光的方法对GAP-43表达位置进行定位。结果 经PCR方法检测得到转基因阳性鼠,Western blotting结果显示 GAP-43蛋白的表达量高于同胞对照组的小鼠且差异有统计学意义,免疫荧光染色结果显示GAP-43阳性反应物弥散分布于大脑皮质和海马中,且特异性存在于神经元中。结论 外源基因GAP-43在小鼠基因组中得到整合并稳定遗传,为神经损伤修复及神经发育的相关研究提供了有价值的动物模型。

关键词: GAP-43, 转基因小鼠, 胚胎显微注射

Abstract:

Objective To establish transgenic mice with over expression of GAP-43 protein, in order to study further on effects and mechanism of GAP-43 in repair of the neural injury. Methods The GAP-43 RNA was extracted from adult C57 mouse and reversely transcripted for obtaining its cDNA. The GAP-43 cDNA was then cloned into the pCEP4-PDGF vector and verified by PCR and sequencing. The plasmid vector pCEP4-PDGF-GAP-43 was transfected into the E. coli DH5α. Transgenic component of pCEP4-PDGF-GAP-43 was therefore constructed and linearized. After purifying,the vector was microinjected into fertilized mouse eggs. These eggs were then transplanted into pseudopregnant mice. The genotype of transgenic mice was identified by PCR. The over expression of GAP-43 protein in the hippocampus of transgenic mice was detected by Western blotting and immunoflourescent staining.Results There were 9 mice showing to have a positive integration of transgenic GAP-43 among 19 experimental mice, by detection of PCR.The Western blotting and immunoflourescence showed that the expression of GAP-43 protein in those transgenic mice was higher than that in the control group. Conclusion The transgenic mouse with GAP-43 over expression was successfully established in the present study.

Key words: GAP-43, transgenic mice, embryo microinjection

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