运用CRISPR/Cas9构建CXCR4基因编辑慢病毒载体
发布时间：2021-03-05 12:04
目的:以CXCR4为靶基因,采用CRISPR/Cas9技术构建可诱导表达基因编辑慢病毒载体系统,并且在MKN-45细胞系验证其基因编辑功能。方法:分别以lenti-cas9-BLAST和PX458质粒为模板,扩增Blasticidin s及T2A-GFP基因片段,凝胶电泳回收后与Lenti-guide-puro质粒重组,获得重组质粒Lenti-guide-BLAST-GFP。针对CXCR4基因EXON2设计并合成三条sg RNA,与Lenti-guide-BLAST-GFP重组获得Lenti-guide-BLAST-GFP-sgRNA质粒,扩增后进行慢病毒载体包装。对pCW-Cas9质粒扩增后进行慢病毒包装。然后将上述两种载体依次感染MKN45细胞,压力筛选培养,经DOX 1μg/ml诱导后,检测MKN-45细胞CXCR4蛋白表达和基因突变水平,并验证CXCR4基因编辑前后MKN-45细胞对SDF-1应答改变。结果:经过测序证实,成功构建了Lenti-guide-BLAST-GFP和Lenti-guide-BLAST-GFP-sgRNA质粒,并成功获得Lenti-guide-BLAST...
【文章来源】：重庆医科大学重庆市
【文章页数】：43 页
【学位级别】：硕士
【部分图文】：
三条gRNA成功克隆至重真核表质粒Lenti-guide-BLAST-GFPFigure1ResultsofSangersequencingconfirmingthatthethreegRNAswereinsertedintothelenti-guide-BLAST-GFPplasmid.
图 2 Western Blot 检测 MKN-45 胞首次病毒感染后 Cas9 表estern blotting to detect doxycycline-induced Cas9 expression in MKN-4infection. DOX(-): without doxycycline induction ; DOX(+): induction wdoxycycline for 48 hours .包装 GFP 标记的 gRNA 慢病毒载体感染慢病毒载体后，经历再次抗菌素压力筛选，荧光显微镜镜检胞表达 GFP，阳性率在 90%以上（图 3）。
图 2 Western Blot 检测 MKN-45 胞首次病毒感染后 Cas9 表Figure 2 Western blotting to detect doxycycline-induced Cas9 expression in MKN-45 cells afterthe first infection. DOX(-): without doxycycline induction ; DOX(+): induction with 1 μg/mldoxycycline for 48 hours .3.3.2 成功包装 GFP 标记的 gRNA 慢病毒载体二次感染慢病毒载体后，经历再次抗菌素压力筛选，荧光显微镜镜检 MKN-45细胞，细胞表达 GFP，阳性率在 90%以上（图 3）。
【参考文献】：
期刊论文
[1]The big bang of genome editing technology: development and application of the CRISPR/Cas9 system in disease animal models[J]. Ming SHAO,Tian-Rui XU,Ce-Shi CHEN. Zoological Research. 2016(04)
[2]CXCR4/SDF-1 axis is involved in lymph node metastasis of gastric carcinoma[J]. Bao-Cheng Zhao,Zhen-Jun Wang,Hua-Chong Ma,Jia-Gang Han,Bo Zhao,Department of General Surgery,Beijing Chaoyang Hospital,Capital Medical University,Beijing 100020,China Wei-Zheng Mao,Department of General Surgery,Qingdao Municipal Hospital,Qingdao University Medical College,Qingdao 266071,Shandong Province,China Hui-Min Xu,Department of General Surgery,Weifang People’s Hospital,Weifang 261041,Shangdong Province,China. World Journal of Gastroenterology. 2011(19)
本文编号：3065169
【文章来源】：重庆医科大学重庆市
【文章页数】：43 页
【学位级别】：硕士
【部分图文】：
三条gRNA成功克隆至重真核表质粒Lenti-guide-BLAST-GFPFigure1ResultsofSangersequencingconfirmingthatthethreegRNAswereinsertedintothelenti-guide-BLAST-GFPplasmid.
图 2 Western Blot 检测 MKN-45 胞首次病毒感染后 Cas9 表estern blotting to detect doxycycline-induced Cas9 expression in MKN-4infection. DOX(-): without doxycycline induction ; DOX(+): induction wdoxycycline for 48 hours .包装 GFP 标记的 gRNA 慢病毒载体感染慢病毒载体后，经历再次抗菌素压力筛选，荧光显微镜镜检胞表达 GFP，阳性率在 90%以上（图 3）。
图 2 Western Blot 检测 MKN-45 胞首次病毒感染后 Cas9 表Figure 2 Western blotting to detect doxycycline-induced Cas9 expression in MKN-45 cells afterthe first infection. DOX(-): without doxycycline induction ; DOX(+): induction with 1 μg/mldoxycycline for 48 hours .3.3.2 成功包装 GFP 标记的 gRNA 慢病毒载体二次感染慢病毒载体后，经历再次抗菌素压力筛选，荧光显微镜镜检 MKN-45细胞，细胞表达 GFP，阳性率在 90%以上（图 3）。
【参考文献】：
期刊论文
[1]The big bang of genome editing technology: development and application of the CRISPR/Cas9 system in disease animal models[J]. Ming SHAO,Tian-Rui XU,Ce-Shi CHEN. Zoological Research. 2016(04)
[2]CXCR4/SDF-1 axis is involved in lymph node metastasis of gastric carcinoma[J]. Bao-Cheng Zhao,Zhen-Jun Wang,Hua-Chong Ma,Jia-Gang Han,Bo Zhao,Department of General Surgery,Beijing Chaoyang Hospital,Capital Medical University,Beijing 100020,China Wei-Zheng Mao,Department of General Surgery,Qingdao Municipal Hospital,Qingdao University Medical College,Qingdao 266071,Shandong Province,China Hui-Min Xu,Department of General Surgery,Weifang People’s Hospital,Weifang 261041,Shangdong Province,China. World Journal of Gastroenterology. 2011(19)
本文编号：3065169
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