成年狨猴肾脏细胞体外培养体系的建立及CRISPR/ Cas9 基因编辑的应用
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(中国医学科学院医学实验动物研究所,北京 100021)

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R-33

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Establishment of an in vitro culture system for adult marmoset kidney cells and applications of clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 gene editing
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(Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) &Peking Union Medical Collage (PUMC), Beijing 100021, China)

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    摘要:

    目的 建立成年狨猴肾脏细胞体外培养的培养体系,并将Cas9 编辑系统初步应用于所培养的狨猴肾脏细胞,建立检测sgRNA 切割活性的体系?方法 取成年的狨猴肾脏,采用贴壁法?消化法得到狨猴肾脏细胞?对细胞进行生长曲线测定?转染试剂(Viafect 和Lipo2000)及抗性(嘌呤霉素和杀稻瘟菌素)浓度筛选,将SIRT1 的sgRNA 质粒和Cas9 质粒共转染狨猴细胞,提取基因组并对基因修饰目的片段扩增,并将扩增产物进行测序?结果建立狨猴肾脏细胞体外培养体系;ViaFect 转染试剂转染率大于60%;细胞抗性筛选适宜浓度嘌呤霉素4 μg/ mL,杀稻瘟菌素8 μg/ mL;测序结果表明,从sgRNA 的PAM 序列附近开始出现突变峰,证明sgRNA 成功引入突变?结论 成功建立了狨猴肾脏细胞的体外培养?细胞转染和抗性筛选体系;CRISPR/ Cas9 可以用于狨猴肾脏细胞编辑,为基因修饰狨猴靶点选择提供依据?

    Abstract:

    Objective To establish an in vitro culture system of adult marmoset kidney cells, and create a meansfor detecting single guide (sg) RNA cutting activity based on the Cas9 editing system. Methods A growth curve wasinitially determined for adult marmoset kidney cells. The growth curve, transfection reagent and resistance concentrationwere determined. These were then used to co-transfect SIRT1 sgRNA and Cas9 into marmoset cells. Following transfection,the genome was extracted and sequenced. Results The transfection rate of ViaFect was greater than 50% ~ 70%. Theoptimum concentration of puromycin was 4 μg/ mL and that of blasticidin was 8 μg/ mL for cell resistance screening.Sequencing result showed that mutation peaks appeared near the PAM sequence of sgRNA, which proved that sgRNA wassuccessfully introduced mutations into the marmoset genome. Conclusions We successfully established an in vitro culture,transfection and resistance screening system for marmoset kidney cells, which can be used as a reference for gene editing site testing. CRISPR/ Cas9 is suitable for renal cell editing in marmosets.

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丛日旭,佟巍,向志光,张丽芳,刘先菊,阮研硕,郭智,刘云波.成年狨猴肾脏细胞体外培养体系的建立及CRISPR/ Cas9 基因编辑的应用[J].中国比较医学杂志,2019,29(8):50~54.

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  • 收稿日期:2019-01-23
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  • 在线发布日期: 2019-09-12
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