摘要
【目的】利用慢病毒介导的CRISPR/Cas9基因组编辑技术获得编辑BMPR-IB(bone morphogenetic protein receptor,type IB)基因的猪胎儿成纤维细胞(pig fetal fibroblasts,PFF),并研究该基因被编辑后对骨形态发生蛋白(bone morphogenetic proteins,BMPs)信号通路中重要功能基因表达的影响。【方法】针对猪的BMPR-IB基因的外显子8,利用在线软件http://crispr.mit.edu获得了21条sg RNAs(single-guide RNAs)序列;得分最高的sg RNA与互补序列(包含接头)退火成双链后连接入线性化的lenti CRISPR v2质粒以获得打靶质粒,打靶质粒与包装质粒ps PAX2和p CMV-VSV-G按5﹕4﹕1质量比混合后通过293T细胞包装慢病毒。慢病毒溶液经0.45μm滤膜回收后与PFF细胞培养液按照1﹕1混合、并加入聚凝胺(polybrene)至终浓度为6μg·m L-1,在1 000 g转速、32℃下离心感染PFF细胞1 h。感染3 d后,细胞在含3.5μg·m L-1嘌呤霉素的培养液中筛选6—7 d,最终获得编辑BMPR-IB基因的PFF细胞克隆群。针对编辑(打靶)细胞,首先通过T7E1酶切检测突变体,初步判断打靶效率,再通过PCR、PCR-TA克隆分析细胞的编辑及脱靶情况。利用RT-PCR检测编辑和对照组细胞中与BMPs信号通路相关的重要基因的表达情况;Western blotting检测编辑细胞与对照组细胞中BMPR-IB基因的蛋白表达量。利用Cell Counting Kit-8(CCK-8)试剂盒检测编辑细胞及对照组细胞的增殖能力。【结果】T7E1酶切及PCR测序均证实PFF细胞成功打靶目标DNA区域;TA克隆测序表明目标区域发生了插入与缺失突变,突变率为70%。针对20个潜在的脱靶位点的TA克隆测序结果表明,仅1个位点出现了10%(2/20)的脱靶情况。RT-PCR结果显示,打靶细胞与对照组细胞相比,BMPR-IB、Cylin D2、Cdk2和Bcl2基因的表达量均极显著下降(P<0.01)。Western blotting结果显示,打靶细胞BMPR-IB基因的表达量较对照组细胞降低62%。CCK-8检测试验表明,同一代细胞中,打靶PFF的增殖能�
【Objective】 The aims of this study were to edit the BMPR-IB gene in pig fetal fibroblasts（PFF） via a lentivirus-mediated CRISPR/Cas9 genome editing technology, and to investigate its effects on expression of relevant functional genes in the bone morphogenetic proteins（BMPs） signaling pathway. 【Method】 Twenty one single-guide RNAs（sg RNAs） targeting the eighth exon of porcine BMPR-IB gene were designed by the online software http：//crispr.mit.edu. The sg RNA sequence with the highest score was selected for annealing with its complementary sequence（including adapters）, and then the double-stranded DNA was ligated into the linearized lenti CRISPR v2, with the aim to obtain targeting plasmid. The targeting plasmid was mixed with packing vectors ps PAX2 and p CMV-VSV-G at 5﹕4﹕1 molar ratio, then used to produce the recombinant lentivirus in 293 T cells. To generate the induction mixture, the lentivirus supernatant was filtered through 0.45 μm, mixed with equal volume of fresh PFF growth medium, and finally polybrene was added to a final concentration of 6 μg·m L-1. PFF cells were infected in induction mixture and centrifuged at 1 000 g for 1 h at 32℃, then cultured in a 37℃ incubator for 3 days. Three days post-transfection cells were selected with 3.5 μg·m L-1 puromycin for 6-7 days, and resistant clones targeting BMPR-IB were expanded. Targeting cells were screened first by T7 E1 digestion, and then the PCR and PCR-TA cloning were performed to confirm correct targeting. Quantitative real-time PCR was performed to detect the expression levels of relevant functional genes in BMPs signaling pathway. Protein expression of the BMPR-IB gene was detected by Western blotting. Cell Counting Kit-8（CCK-8） kit was used to measure the proliferation capacity of targeted cells and control group cells. 【Result】 Both T7 E1 assay and PCR sequencing showed that the targeted region was successfully edited in targeted cells. TA cloning and sequencing revealed the desired insertion and delet
作者
杨强
徐盼
蒋凯
乔传民
任军
黄路生
幸宇云
YANG Qiang;XU Pan;JIANG Kai;QIAO ChuanMin;REN Jun;HUANG LuSheng;XING YuYun(State Key Laboratory of Pig Genetic Improvement and Production Technology, Jiangxi Agricultural University, Nanchang 33004)
出处
《中国农业科学》
CAS
CSCD
北大核心
2018年第7期1378-1389,共12页
Scientia Agricultura Sinica
基金
国家自然科学基金(31560304)
国家科技重大专项(2016ZX08006-003)
