人/猪OSBPL2同源性比较及猪PFFs靶基因敲除细胞系的建立
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国家自然科学基金(81771000,31571302);江苏省重点研发计划(社会发展)项目(BE2016762)


Homology comparison between human and pig OSBPL2 and establishment of pig PFFs with target gene knockout
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    摘要:

    目的:基于CRISPR/Cas9技术构建猪胎儿成纤维细胞(porcine fetal fibroblasts,PFFs)OSBPL2敲除细胞系,为构建小型猪致聋基因缺陷动物模型奠定重要的前期工作基础。方法:首先通过生物信息学方法对人与猪OSBPL2基因共线性和同源性进行分析,预测并模拟人与猪OSBPL2蛋白质二级、三级结构。其次,设计合成靶向猪OSBPL2第5、6外显子设计单导向RNA(single guide RNA,sgRNA),以pX330质粒为载体,构建含有Cas9骨架的重组载体,转染至猪PFFs中,G418药物筛选阳性单克隆细胞。最后,T7EN1酶切实验检测靶向效率,序列分析检测单克隆细胞基因型。结果:生物信息学分析结果表明人与猪的OSBPL2在染色体上具有较好的共线性关系,蛋白质氨基酸同源性高达88%,且具有相似的功能结构域。成功构建打靶OSBPL2基因的Cas9/sgRNA表达载体,转染PFFs细胞,药物筛选获得OSBPL2基因双敲的单细胞克隆并测序证实了基因突变型。结论:人和猪OSBPL2基因具有高度的同源性。构建成功的Cas9/sgRNA表达载体在PFFs中预期实现了OSBPL2基因编辑并获得基因双敲的单细胞克隆,为后续OSBPL2基因敲除猪模型构建提供了必需的实验材料。

    Abstract:

    Objective:To build OSBPL2-knockout porcine fetal fibroblasts(PFFs)based on CRISPR/Cas9 technology and lay the important preparative foundation for establishing an experimental animal model of deafness in miniature pigs with OSBPL2 defects. Methods:Bioinformatics methods were applied to analyze the collinearity and homology of OSBPL2 between human and pig. The secondary and tertiary structures of OSBPL2 protein of human and pig were predicted and simulated. Two single-guide RNAs(sgRNAs)respectively targeting the fifth and the sixth exon of pig OSBPL2 were designed,synthesized and cloned into pX330 plasmid. G418 was used to obtain the positive monoclonal cells after transfection into PFFs. Finally,the efficiency of CRISPR/Cas9 mediated knockout was assessed by the T7EN1 enzyme digestion assay and the genotypes of monoclonal cells were identified by sequencing analysis. Results:Bioinformatic analysis revealed that the OSBPL2 of human and pig had a good collinearity on chromosome,highly homologous amino acid sequence(88%)and similar functional domain characteristics. CRISPR/Cas9 expression vectors targeting OSBPL2 were constructed and transfected into PFFs. OSBPL2-knockout monoclonal cells with mutant genotypes were obtained by drug screening and confirmed by DNA sequencing. Conclusion:The human and pig OSBPL2 sequences and their protein structures are highly homologous. CRISPR/Cas9 expression vectors were constructed to achieve OSBPL2 gene targeting in PFFs. OSBPL2-knockout monoclonal cells were obtained,which could contribute to the construction of OSBPL2-knockout miniature pig model.

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曾华沙,姚 俊,王红顺,王 盈,杨海元,曹 新,戴一凡.人/猪OSBPL2同源性比较及猪PFFs靶基因敲除细胞系的建立[J].南京医科大学学报(自然科学版),2018,(2):149-154

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  • 收稿日期:2017-04-16
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  • 在线发布日期: 2018-03-02
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