CRISPR-Cas9敲减鹅硬脂酰辅酶A去饱和酶基因的慢病毒质粒构建

Construction of a CRISPR-Cas9 knockdown lentiviral plasmid of goose(Anas platyrhynchos)stearoyl-coenzyme A desaturase gene

为进一步探究鹅卵泡颗粒细胞内源性脂肪酸合成代谢机制,利用CRISPR-Cas9技术构建靶向鹅硬脂酰辅酶A去饱和酶(stearoyl-coenzyme A desaturase,SCD)基因的慢病毒敲减质粒并进行慢病毒包装。首先设计鹅SCD基因的单链指导RNA(single-guide RNA,sgRNA)序列,其次体外合成并验证裂解效率,最后利用psPAX2和pMD2.G包装质粒制备psgRNA-mCherry-T2A-Puro和pLenti-Cas9-T2A-EGFP慢病毒质粒。结果表明,慢病毒质粒被成功构建,且其感染中国仓鼠卵巢(Chinese hamster ovary,CHO)细胞后筛选出能同时表达红色荧光蛋白和增强绿色荧光蛋白的强双阳性细胞群。这为后续感染鹅原代颗粒细胞并敲减其SCD基因研究奠定了基础。

In order to further explore the mechanism of endogenous fatty acid synthesis and metabolism in goose granulosa cells,we used CRISPR-Cas9 technology to construct knockdown plasmids of a goose targeted stearoyl-coenzyme A desaturase(SCD)gene and package lentivirus.First,we designed the sequence of singleguide RNA(sgRNA)of goose SCD gene;second,synthesized in vitro and tested the lysis efficacy of sgRNA to target DNA site by endonuclease cleavage assays;finally,prepared the lentiviral plasmid of psgRNA-mCherry-T2A-Puro and pLenti-Cas9-T2A-EGFP using psPAX2 and pMD2.G as package plasmids.Results showed that the lentiviral plasmid was successfully constructed,and the strong double positive cell groups co-expressing the red fluorescent protein(mCherry)and enhanced green fluorescent protein(EGFP)were screened out when the lentiviral plasmid infected the Chinese hamster ovary(CHO)cells.The above results lay the foundation for infecting goose primary granulosa cells and targeting knockdown the SCD gene.