人/猪SERPING1同源性比较及猪SERPING1敲除细胞系的建立

Homology comparison between human and pig SERPING1and establishment of pig PFFs with SERPING1 knockout

目的利用CRISPR/Cas9基因编辑技术建立SERPING1-/-猪胎儿成纤维细胞(PFFs)系,为构建遗传性血管性水肿模型提供细胞实验材料。方法首先比较人/猪SERPING1氨基酸同源性。其次,分析猪SERPING1基因有效的编码区,据此选择第八外显子为敲除靶点,设计并合成靶向猪SERPING1的单导向RNA(sgRNA),连接到含有Cas9核酸内切酶的pX330载体上,构建SERPING1打靶载体pX330sgRNA,将其转染至猪胎儿成纤维细胞中,G418药物筛选阳性单克隆细胞。最后,用T7E1酶切实验检测靶点编辑情况,测序验证单克隆细胞基因型。结果生物信息学分析结果提示人/猪SERPING1蛋白氨基酸同源性较高,氨基酸比对相似性达到65.87%。成功构建打靶SERPING1的载体,并转染到细胞,药物筛选获得SERPING1基因敲除的单克隆细胞,测序确认了突变的基因型。结论人/猪SERPING1蛋白同源性较高,适合用于构建遗传性血管性水肿模型。构建的Cas9/sgRNA表达载体实现了SERPING1基因编辑,获得基因敲除的单细胞克隆,为后续SERPING1-/-猪模型的构建提供了必需的实验材料。

Objective To build SERPING1-knockout porcine fetal fibroblasts(PFFs)based on CRISPR/Cas9 technology and provide cell experimental materials for the construction of hereditary angioedema models.Methods Firstly,protein structure prediction software was used to analyze the amino acid homology between human and pig SERPING1.Secondly,the eighth exon was selected as the knockout target by screening the effective coding region of the pig SERPING1 gene.A single guide RNA targeting pig SERPING1 was designed,synthesized and linked to pX330 vector containing Cas9 endonuclease.G418 was used to obtain the positive monoclonal cells after transfection into PFFs.Finally,the circumstance 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 SERPING1 protein of human and pig had high homology,amino acid sequence identity reached 65.87%.A vector targeting SERPING1 was constructed successfully and transfected into cells.Monoclonal cells with knockout SERPING1 gene were obtained through drug screening.Sequencing confirmed the mutant genotype.Conclusion The human and pig SERPING1 sequences and their protein structures are highly homologous,and it is suitable for the construction of disease model.CRISPR/Cas9 expression vectors were constructed to achieve the SERPING1 gene targeting in PFFs.SERPING1-knockout monoclonal cells were obtained,which could contribute to the construction of the SERPING1 knockout miniature pig model.