摘要
目的:构建靶向活化转录因子3(ATF3)在CRISPR/Cas9技术编辑的慢病毒载体。方法:针对ATF3基因设计并合成三条对应的向导(gRNA)序列和一条无意义序列,向导RNA序列与双酶切U6-EF1a-Cas9-FLAG-P2A-EGFP载体链接,DNA测序鉴定重组载体。荧光/绝对定量法测定慢病毒浓度。共转染人微血管静脉内皮细胞(HMEC)后,实时定量荧光PCR检测ATF3mRNA表达,验证HMEC细胞中是否含有ATF3基因及其是否有突变。对照病毒和三个靶点慢病毒分别感染Cas9蛋白稳定株,感染后混合克隆进行抽提基因组DNA,将得到PCR产物进行错配酶法进行酶切,对于筛靶中阳性的PCR产物一组序列与野生型对比。Westernblot检测ATF3蛋白表达,验证转染效果。定量PCR检测ATF3mRNA的改变。结果:测序表明ATF3在Cas9编辑下慢病毒载体构建成功。HMEC倒置荧光显微镜荧光视野和明视野进行对比,感染可达到80%转染率,GFP持续稳定表达。Westernblot结果显示,ATF3蛋白表达有明显下调(P<0.05)。结论:本研究成功构建了ATF3Cas9编辑慢病毒载体,为进一步研究ATF3在静脉桥血管再狭窄中早期内皮功能失调作用机制及基因治疗提供了实验依据。
Objective: To construct activating transcription factor 3 ( ATF3) CRISPR/CAS9 lentiviral vector. Methods: One Three corresponding guide RNAs ( gRNA) sequences and one nonsense sequence were designed and synthesized for ATF3 gene. The guide RNA sequence was linked with enzyme cleavage carrier of U6-EF1a-Cas9-FLAG-P2A-EGFP,and the recombinant carriers was identified by DNA sequencing. The lentivirus concentration was determined by fluorescence /absolute quantification.After co-transfection of human microvascular endothelial cells ( HMEC),real-time quantitative fluorescent PCR was used to detect ATF3 mRNA expression,and it was confirmed whether HMEC cells contained ATF3 gene and whether it had mutations. The control virus and the three target lentiviruses respectively infect the Cas9 protein stable strain,and the mixed clones after infection are used to extract genomic DNA,and the PCR product is subjected to mismatch enzyme digestion,and a set of sequences of positive PCR products in the sieve target is obtained. Contrast with wild type. The expression of ATF3 protein was detected by Western blot,and the editing effect was verified. Results: Sequencing showed that ATF3 was successfully constructed under the editing of Cas9. The fluorescence field of HMEC inverted fluorescence microscope was compared with the bright field. The infection could reach 80% transfection rate and GFP continued to express stably. Western blot result showed that ATF3 protein expression was significantly down-regulated ( P<0. 05). Conclusions: This study successfully constructed the targeting of ATF3Cas9 editing lentiviral vector,which provides an experimental basis for further study of the mechanism of early endothelial dysfunction and gene therapy of ATF3 in venous vascular graft.
作者
周绍酉
周宁
张魁
李扬
赵洋
宋邦荣
郑居兵
刘涛帅
董然
ZHOU Shaoyou;ZHOU Ning;ZHANG Kui;LI Yang;ZHAO;Yang;SONG Bangrong;ZHENG Jubing;LIU Taoshuai(Department of Cardiac Surgery,Beijing Anzhen Hospital,Capital Medical University,Beijing Institute of Heart,Lung and Blood Vessel Diseases,Beijing 100029,China)
出处
《心肺血管病杂志》
2019年第7期799-804,共6页
Journal of Cardiovascular and Pulmonary Diseases
基金
国家自然科学基金资助项目(8177020427)
