利用锌指核酸酶(ZFN)技术介导敲除猪卵母细胞孤雌胚胎中肌肉生长抑制素基因(MSTN)的初步研究  

Study on Myostatin Gene(MSTN) Knockout of Porcine(Sus scrofa) Oocyte Parthenogenetic Embryo by Zinc-finger Nucleases(ZFN)

作　　者：刘杰[1] 高鹏飞[1,2] 崔文涛[2] 李奎[2] 

机构地区：[1]青岛科技大学生物工程与技术系,青岛266042 [2]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《农业生物技术学报》2014年第4期464-469,共6页Journal of Agricultural Biotechnology

基　　金：基本科研业务费专项资金项目(No.2014ywf-yb-10)

摘　　要：肌肉生长抑制素基因(myostatin,MSTN)是转化生长因子-β(transforming growth factor,TGF-β)超家族的一员,具有特异性调控动物肌肉生长的功能。为证明锌指核酸酶(zinc-finger nucleases,ZFN)修饰技术在猪(Sus scrofa)胚胎水平上对MSTN基因修饰的效率,筛选高效的作用靶点,本研究通过构建针对MSTN基因特异位点的ZFN质粒,采用显微注射方法,将其注射进入猪卵母细胞孤雌胚胎细胞,采用PCR对培养后的囊胚进行MSTN基因的扩增检测,并对其进行序列测定比较分析。研究结果表明,373枚注射囊胚基因组经MSTN引物特异性扩增并测序,检测到2枚发生突变,均在ZFN靶位点处,第39号囊胚发生14个碱基的单碱基突变,第321号囊胚发生11个碱基的单碱基突变,ZFN介导的打靶效率为0.54%。本研究进一步验证了ZFN基因编辑技术在敲除猪MSTN基因上应用的可行性,为MSTN基因敲除猪的制备提供了依据。As a member of transforming growth factor（TGF-β）, myostatin（MSTN） plays an important role for the specific regulation of muscle growth of animals. The purpose of this study is to verify the efficiency of zinc-finger nucleases（ZFN） gene editing on porcine（Sus scrofa） and to pick out the efficient gene whicn includes targeting sites. Using the method of microinjection, ZFN plasmid were injected that were specifically targeted on MSTN into porcine oocyte parthenogenetic embryo . Then the MSTN sequence of cultured embryos were amplified and analyzed by PCR. The results showed that totally 373 embryos were injected, and 2 mutate sequences were found after the specifically amplification by MSTN primers which disigned according JN630464（GenBank accession number）, 14 single base mutations were found in No. 39 embryo, and 11 single base mutations were found in No. 321 embryo. Both mutations occurred in ZFN targeting sites, the targeting efficiency was about 0.54%. The results ensure the possibility and provide feasibility analysis for the preparation of MSTN knockout pig by ZFN gene editing technology.

关 键 词：锌指核酸酶技术 肌肉生长抑制素基因 显微注射 猪卵母细胞孤雌胚胎 

分 类 号：S828.2[农业科学—畜牧学]