摘要
为了研究肌肉生长抑制素(MSTN)的相关功能,应用分子生物学方法构建敲除MSTN的AAVSaCas9载体,通过转染293T细胞提取基因组后进行T7酶切鉴定、TA克隆及测序确定该基因的敲除效果;将鉴定正确的AAV-SaCas9重组质粒与pHelper质粒共转染AAV-293细胞3 d后,分离纯化病毒并用实时荧光定量PCR法检测病毒滴度。结果显示,成功构建了敲除MSTN基因的AAV-SaCas9重组载体,T7酶切和测序鉴定出sgRNA2位点可以对MSTN进行编辑,并成功将其包装成病毒,经荧光定量PCR鉴定病毒滴度为2.73×10^(12)vg/mL。
To study the function of myostatin（ MSTN）,molecular biology method was applied to construct the recombinant vector of MSTN knockout AAV-SaCas9,the transfected 293T cells were extracted for the genome and identified by T7 endonuclease. Then the mutations were further confirmed by TA cloning and sequencing. The AAV-293 cells were co-transfected with AAV-SaCas9 and helper plasmids. Virus was isolated and purified after three days,and its titer was determined by real-time fluorescence quantitative PCR. Our results showed that the recombinant vector of MSTN knockout AAV-SaCas9 was successfully constructed. MSTN could be edited by sgRNA2 identified by T7 digestion and sequencing,and AAV-SaCas9 was successfully packaged into virus. Finally the titer was 2. 73 × 10^（12） vg/mL,which was identified by fluorescence quantitative PCR.
出处
《河南农业科学》
CSCD
北大核心
2018年第1期118-121,共4页
Journal of Henan Agricultural Sciences
基金
河南省科技计划项目(142300413209)
农业部转基因重大专项(2014ZX0801015B)
