Generation of B Cell-Deficient Pigs by Highly Efficient CRISPR/Cas9-Mediated Gene Targeting
Generation of B Cell-Deficient Pigs by Highly Efficient CRISPR/Cas9-Mediated Gene Targeting
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DOI：
10.1016/j.jgg.2015.05.002
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发表时间：
2015-08-20
影响因子：
5.9
通讯作者：
Dai, Yifan
中科院分区：
文献类型：
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作者：
Chen, Fengjiao;Wang, Ying;Dai, Yifan
产生B细胞缺陷突变体是在大型动物模型中产生人类抗体库的第一步。在本研究中，我们应用聚类规则间隔短回文重复(CRISPR)/CRISPR相关（Cas）系统靶向猪IgM重链基因的JH区域，该区域对B细胞的发育和分化至关重要。在原代猪胎成纤维细胞（PFFs）中转染igm靶向Cas9质粒，可诱导高达53.3%的菌落发生基因敲除（KO），其中四分之一的菌落存在双等位基因修饰，这远远高于传统的同源重组（HR）。利用体细胞核移植（SCNT）技术，培育了3头双等位基因IgM重链基因突变仔猪。仔猪没有产生抗体的B细胞，这表明IgM重链基因的双等位基因突变有效地敲除了IgM的功能，导致了B细胞缺陷表型。我们的研究表明，CRISPR/Cas9系统结合SCNT技术是一种有效的猪基因组编辑方法。
Generating B cell-deficient mutant is the first step to produce human antibody repertoires in large animal models. In this study, we applied the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system to target the JH region of the pig IgM heavy chain gene which is crucial for B cell development and differentiation. Transfection of IgM-targeting Cas9 plasmid in primary porcine fetal fibroblasts (PFFs) enabled inducing gene knock out (KO) in up to 53.3% of colonies analyzed, a quarter of which harbored biallelic modification, which was much higher than that of the traditional homologous recombination (HR). With the aid of somatic cell nuclear transfer (SCNT) technology, three piglets with the biallelic IgM heavy chain gene mutation were produced. The piglets showed no antibody-producing B cells which indicated that the biallelic mutation of the IgM heavy chain gene effectively knocked out the function of the IgM and resulted in a B cell-deficient phenotype. Our study suggests that the CRISPR/Cas9 system combined with SCNT technology is an efficient genome-editing approach in pigs.