| 王 敏1，黄 翔1，石 翾1，刘小凤1，曾检华2，刘小红1，陈瑶生1，何祖勇1.应用CRISPR/Cas9 编辑广东
小耳花猪MSTN 基因[J].广东农业科学,2017,44(2):141-148 |
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应用CRISPR/Cas9 编辑广东
小耳花猪MSTN 基因 |
| Editing MSTN gene of Guangdong Xiaoerhuapig by using CRISPR/Cas9 |
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| DOI：10.16768/j.issn.1004-874X.2017.02.022 |
| 中文关键词: MSTN CIRSPR/Cas9 广东小耳花猪 基因编辑 |
| 英文关键词: MSTN CRISPR/Cas9 Guangdong Xiaoerhua pig gene editing |
| 基金项目:国家转基因生物新品种培育重大专项（2016ZX08006003-006）；广东省自然科学基金（2016A0
30313310）；广东省扬帆计划项目（2014YT02H042） |
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| 摘要点击次数: 2768 |
| 全文下载次数: 1286 |
| 中文摘要: |
| MSTN 基因是肌肉生长抑制基因，破坏MSTN 基因的表达成为提高畜禽肌肉产量的有效途径。
通过应用CRISRP/Cas9 编辑技术对广东小耳花猪MSTN 基因的外显子区域进行编辑，破坏MSTN 基因的读
码框来抑制其表达，从而提高广东小耳花猪的肌肉产量。共设计了6 条靶向MSTN 基因3 个不同外显子的
gRNA，经过T7E1 酶切检测和TA 克隆分析，发现其中3 条gRNA 能够发生有效切割，其中切割效率最高的
达28.3%，最低为17.0%。gRNA1-1 和gRNA3-3 突变的广东小耳花猪PEF 细胞系，突变效率均在50% 以
上，其中三号外显子上突变位点的突变率达到61.5%。试验结果为后续进行体细胞核移植，生产MSTN 基因
编辑的广东小耳花猪奠定了基础。 |
| 英文摘要: |
| MSTN（ Myostatin） gene functions as a negative regulator of muscle growth. Therefore，disrupting
the expression of MSTN has been proved as an efficient strategy to improve the muscle mass in livestock and poultry.
In this study，we edited the exons of MSTN gene to disrupt its expression in order to increase muscle production of
Guangdong Xiaoerhua pig by using the cutting-edge genome editing technology—CRISPR/Cas9. We designed six
guide RNAs targeting on loci across three exons of MSTN gene. Through T7E1 assay and TA cloning analysis，we
found that three guide RNAs were capable of targeted cutting on MSTN gene efficiently， with the targeting efficiency
ranging from 17.0% to 28.3%. The editing efficiency of gRNA1-1 and gRNA3-3 in PEF cells of Guangdong
Xiaoerhua pig，were both up to 50%. Particularly，editing efficiency in the third exon was as high as 61.5%. This
study provides basis to generate MSTN-edited Xiaoerhua pigs by somatic cell nuclease transfer（SCNT） in future. |
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