锌指核酸酶(ZFN)介导的牛肌肉抑制素基因(MSTN)突变的研究  

Study on the Mutation of Bovine(Bos taurus) Myostatin Gene(MSTN)Using Zinc Finger Nuclease(ZFN)

作　　者：赵宇航[1] 云亭[1] 李荣凤[2] 李雪玲[1] 

机构地区：[1]内蒙古大学教育部哺乳动物生殖生物学及生物技术重点实验室,呼和浩特010021 [2]南京医科大学,南京210029

出　　处：《农业生物技术学报》2014年第10期1204-1212,共9页Journal of Agricultural Biotechnology

基　　金：国家转基因生物新品中培育重大专项(No.2011ZX08010-001);内蒙古大学高层次人才引进科研项目(No.30105-125129)

摘　　要：肌肉生成抑制素(myostatin,MSTN)是肌肉发育的负调控基因,突变后会引起肌肉的过度发育,在肉用动物育种中有非常重要的价值。本研究利用锌指核酸酶(zinc finger nuclease,ZFN)对牛(Bos taurus)MSTN基因的第二外显子特异性剪切,诱导该基因的突变,从而达到敲除该基因的目的。首先对脂质体介导的双质粒共转染牛成纤维细胞的条件进行优化,获得的共转染效率高达19.27%;之后利用两组ZFNs对牛成纤维细胞进行转染,单细胞通过口吸法移入96孔板,经扩大培养后进行PCR筛选,获得18株MSTN基因突变细胞株,其中包括一株双等位基因敲除的细胞系,两组ZFNs对牛成纤维细胞的突变效率分别为6.05%和3.84%;最后,挑选一株突变细胞系进行体细胞核移植研究,共获得24枚重构胚;将去除透明带的重构胚培养于2i培养液中,获得囊胚来源的类滋养层干细胞,通过PCR和测序检测,证明该细胞系在MSTN基因第二外显子具有与供体细胞相同的突变。结果表明,所合成的两组ZFNs可以在MSTN作用位点引起突变,具有很高的突变效率,而且能获得双等位基因突变的细胞株,获得的细胞株能够用作体细胞核移植的供体细胞。本研究为肉用牛的品质改良和育种工作提供了基础资料。Myostatin gene（MSTN） is a negative skeleton muscle development control gene,and mutation of this gene will induce the over development of skeleton muscles,which is extremely valuable for the breeding of the meat animals.In this study,two pairs of zinc finger nucleases （ZFN） aimed at bovine （Bos taurus） MSTN second exon were used to introduce the mutation in bovine MSTN gene to achieve the purpose of knocking out this gene.Firstly,the transfection condition by two plasmids to bovine fibroblasts via lipofectamine was optimized using different combination of DNA and transfection reagent,and the highest transfection efficiency was 19.25％.Secondly,the bovine fibroblasts were transfected with two pair of ZFNs,respectively; single cells were picked to 96-well plate by mouth-pipetting.After PCR screening,eighteen MSTN mutated cell lines were obtained,which included one double allele mutated cell line.The mutation efficiencies of these two pairs of ZFNs were 6.05％ and 3.84％,respectively.Finally,one of the mutated cell lines was used as the donor cells for somatic cell nucleus transfer （SCNT）,and 24 reconstructed blastocysts were obtained.The trophoblast stem-like cells were derived after the zona pellucida removed and cultured in 2i medium.They had the same mutation at MSTN locus with the donor cells according to the PCR and sequencing analysis.In conclusion,ZFNs were very efficient to introduce the mutation to bovine MSTN gene in fibroblasts,and they had the ability to generate double allele mutated cells in one experiment,and the mutated cells were capable to generate the reconstructed embryos for transfer after SCNT.This study provides basic data for the breeding of the beef cattle by MSTN gene mutation.

关 键 词：肌肉生成抑制素(MSTN) 转染效率 锌指核酸酶(ZFN) 突变 体细胞核移植 

分 类 号：S813[农业科学—畜牧学]