AAV-SaCas9敲除MSTN基因病毒的构建及鉴定
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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.
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.
引文
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[7]Woo J W,Kim J,Kwon S I,et al.DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins[J].Nat Biotechnol,2015,33(11):1162-1164.
[8]Price A A,Sampson T R,Ratner H K,et al.Cas9-mediated targeting of viral RNA in eukaryotic cells[J].Proc Natl Acad Sci,2015,112(19):6164-6169.
[9]Jiang W,Bikard D,Cox D,et al.RNA-guided editing of bacterial genomes using CRISPR-Cas systems[J].Nat Biotechnol,2013,31(3):233-239.
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[12]Sun J Y,Anand-Jawa V,Chatterjee S,et al.Immune responses to adeno-associated virus and its recombinant vectors[J].Gene Ther,2003,10(11):964-976.
[13]Ge X,Xi H,Yang F,et al.CRISPR/Cas9-AAV mediated knock-in at NRL locus in human embryonic stem cells[J].Mol Ther Nucleic Acids,2016,5(11):e393.
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[17]Li L B,Chang K H,Wang P R,et al.Trisomy correction in down syndrome induced pluripotent stem cells[J].Cell Stem Cell,2012,11:615-619.
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[19]王顺红,王善禾,蒋可人,等.鸡miR-1749生物信息学分析及真核表达载体构建[J].河南农业科学,2015,44(5):129-133.