一种表达eGFP的rAAV-SaCas9系统的构建及应用  被引量:1

Construction and application of a rAAV-SaCas9 system expressing eGFP

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作  者:赵忆甜 李秀青 王晓帆 高峰[1] 张坤朋[2] 翁少亭 ZHAO Yitian;LI Xiuqing;WANG Xiaofan;GAO Feng;ZHANG Kunpeng;WENG Shaoting(College of Veterinary Medicine,Henan Agricultural University,University,Zhengzhou 450046,China;College of Life and Food Engineering,Anyang Institute of Technology,Anyang Henan 455000,China)

机构地区:[1]河南农业大学动物医学院,河南郑州450046 [2]安阳工学院生命与食品工程学院,河南安阳455000

出  处:《中国兽医学报》2021年第10期1990-1997,共8页Chinese Journal of Veterinary Science

基  金:国家转基因生物新品种培育重大专项资助项目(2016ZX08006001-006);河南省特色优势学科资助项目(203/18xk0102)。

摘  要:目前,CRISPR/Cas基因编辑技术在培育转基因家畜中已经被广泛应用,但是转基因家畜还存在抗病力弱、组织易癌变及功能紊乱等缺点。因此,基因编辑成年家畜的特定组织成为提升家畜生产性能并且避免转基因动物缺点的一种有效方法。本研究构建了一个rAAV9-SaCas9-eGFP系统,包括由EF1α启动子控制的SaCas9和tRNA启动子控制的sgRNA,还包括绿色荧光蛋白eGFP表达盒,通过表达的eGFP能够明确rAAV9-SaCas9-eGFP在细胞中的存在时间以及在组织中的分布。将rAAV9-SaCas9-eGFP系统导入到C57BL/10小鼠大腿肌肉中对肌生成抑制素基因进行编辑,从而改善肌肉质量。结果表明,成功构建了能够表达eGFP荧光蛋白和进行基因编辑的rAAV9-SaCas9-eGFP系统,该系统只在注射的特定位点有eGFP荧光的表达,没有扩散到周围组织中,这对避免非靶向组织基因编辑以及突变有重要意义。rAAV9-SaCas9-eGFP系统在C57BL/10小鼠大腿肌肉中对肌生成抑制素基因有较高编辑效率,能够明显增加肌肉质量和肌细胞体积,在提高肉用家畜的生产性能方面具有潜在的应用价值。At present,CRISPR/Cas gene editing technology has been widely used in breeding transgenic livestock.However,transgenic livestock have some disadvantages such as weak disease resistance,tissue cancerization and functional disorder.Therefore,gene editing of specific tissues of adult livestock has become an effective way to improve livestock performance and avoid the disadvantages of transgenic animals.In this study,we constructed a rAAV9-SaCas9-eGFP system,which included SaCas9 controlled by EF1αpromoter and sgRNA controlled by tRNA promoter.In addition,eGFP gene was inserted into the system,and the expression level of rAAV9-SaCas9-eGFP in cells and the diffusion range in tissues were determined by the fluorescence of eGFP.Then,we introduced the rAAV9-SaCas9-eGFP system into the thigh muscle of C57 BL/10 mice to edit the myostatin gene and improve the muscle mass.The results showed that we successfully constructed the rAAV9-SaCas9-eGFP system which could express eGFP fluorescent protein and perform gene editing,and the eGFP was only expressed at the specific injection site,and did not spread to the surrounding tissues,which has important implications for avoiding gene editing and mutations in untargeted tissues.Moreover,the rAAV9-SaCas9-eGFP system showed high editing efficiency for myostatin gene in thigh muscle of C57 BL/10 mice,and could significantly increase muscle mass and muscle cell volume.It has potential application value to improve the production performance of meat livestock.

关 键 词:基因编辑 rAAV-SaCas9系统 肌肉生长抑制素 肌肉注射 成肌细胞 

分 类 号:S852.65[农业科学—基础兽医学] Q78[农业科学—兽医学]

 

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