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.pdfVIP

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㈣珊的初步研究 刘杰1高鹏飞“2崔文涛2’李奎2 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 +通讯作者，cMl224@126．com 摘 要 肌肉生长抑制素基因(myostatin，椰Z聊是转化生长因子一D(仃ansfo硼ing growth factor，TGF．p)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(S淞sc嘲)胚胎水平上对髑删基因修饰的效率，筛选高效的作用靶点，本研究通过构建针对 椰删基因特异位点的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椰刑基因的扩增检测，并对其进行序列测定比较分析。研究结果表明，373枚注射 囊胚基因组经鹏删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椰删基因上应用的可行性，为椰州基因敲除猪的制备 提供了依据。 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 Study on Myostatin Gene㈣嬲，)Khockout of Porcine(S邯scrl咖) Oocyte Parthenogenetic Emb巧o by Zinc—finger Nucleases(ZFN) LIUJiel GA0 Peng—Feil，2 CUI Wen—Ta02’ LI Kui2 l Depanment of Bioengineering aIld Biotechnology，C01lege of Chemical Engineering，Qingdao University of Science＆1’echnology，Qingdao 266042，china；2Institu￡eofAnimal science，chineseAcademyofA珈cultl工ral sciences，Be妇ing 100193，china 奉Corresoponding authorc叭1224@126．com Abstract As a member of自ransfomling growth factor(TGF-p)，myostatin㈣刀、，)plays an important r01e for the speci6c regulation of muscle growth of animals．The pu印ose of this study is to Veri矽the e伍ciency of zinc—finger nucleases(zFN)gene editing on porcine(S嬲scrQ向)and to pick out the efficient gene whicn includes ta唱eting sites．Using the method of microi巧ection，zFN plasmid were injected that were specifically targeted on鹏州into porcine ooc”e panhenogenetic embWo．Then the椰删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 aRer the specifically砌plification by邶刑primers which disigIled according Ⅲ630464(GenBaIlk accession肌mber)，14 single base mutations were found in No．39 emb叮o，and 1l single base mutations were found in No．32 1 embUo．Both mutations occurred in ZFN ta唱eting sites，the ta谱eting e伍ciency was about 0．54％．The results ensure the possibili够and provide feasibil