标题：SD大鼠超排及TALEN质粒显微注射构建基因敲除大鼠

      作者：卞洲艳，杨 政，徐 蔓，张洁钰，廖海含，唐其柱
    (武汉大学人民医院心内科 武汉大学心血管病研究所，湖北 武汉 430060)

      卷次： 2013年24卷21期

      【摘要】 目的 应用TALEN技术构建基因敲除大鼠，并探讨SD大鼠超数排卵、显微注射浓度、胚胎移植方
式对构建大鼠出生率、阳性率的影响。方法 将SD雌鼠超数排卵后，取受精卵显微注射由TALEN质粒体外转
录成的mRNA，注射后存活的受精卵输卵管移植，观察大鼠的出生率，并测序判定出生大鼠的基因敲除阳性率。
结果 4~5周和5~6周的雌鼠超数排卵见栓率[(74±9)% vs (77±6%)%]、取卵数量[(25±2)枚vs (23±2)枚]以及卵的
受精率[(85±2)% vs (93±2)%]差异均无统计学意义(P＞0.05)；结扎公鼠和受体雌鼠在 8~10周时见栓率最高
[(23.02±5.26%)%]，使用12周后见栓率明显下降[(11.66±2.40)%]；单侧和双侧输卵管移植大鼠出生率差异无统计
学意义(37.88% vs 36.49%)，但单侧移植大鼠存活率高于双侧移植(37.88% vs 20.27%，P<0.05)，且单细胞移植大鼠
出生率显著高于双细胞移植。质粒注射浓度在20 ng/?l组大鼠基因敲除阳性率最高(7.5%)。结论 应用TALEN
技术成功构建基因敲除大鼠，显微注射质粒浓度20 ng/?l时大鼠出生率不受影响且基因敲除阳性率最高。

      【关键词】 Sprague-Dawley大鼠；TALEN；显微注射；敲除

      【中图分类号】 R-332 【文献标识码】 A 【文章编号】 1003—6350（2013）21—3121—05


Knockout rats construction by superovulation of SD rats and embryo microinjection of TALENs.
BIAN Zhou-yan,
YANG Zheng, XU Man, ZHANG Jie-yu, LIAO Hai-han, TANG Qi-zhu. Department of Cardiology, People's Hospital of Wuhan
University, Cardiovascular Research Institute of Wuhan University, Wuhan 430060, Hubei, CHINA

【Abstract】 Objective To construct knockout rats by embryo microinjection of transcription activator-like ef-
fector nucleases (TALENs), and investigate the effect of superovulation, microinjection and embryo transplantation on
birth rate and positive rate of rats. Methods Rat zygotes obtained from superovulation were oviductal transferred af-
ter microinjection of the mRNA which was got from the in vitro transcription of TALEN plasmid. Results The posi-
tive rate of pseudo-pregnant female rat was high when the vasectomized rats and recipient rats were 8-10 weeks old,
and decreased obviously after mating for 12 weeks. The birth rate had no difference between unilateral and bilateral
oviduct transplantation, but survival rate of unilateral oviduct transplantation was significantly higher than that of bilat-
eral oviduct transplantation. And the positive rate of knockout rats was highest in the group of 20 ng/?l microinjection
of TALENs. Conclusion The 20 ng/?l microinjection of TALENs was the best level to get the highest positive rate
of knockout rats without the effect on birth rate.

      【Key words】 Sprague-Dawley rat; Transcription activator-like effector nucleases; Microinjection; Knockout
基金项目：国家自然科学基金青年项目(编号：81000036)；湖北省科技条件平台建设专项(编号：302-132091)