目的:以CXCR4為靶基因,采用CRISPR/Cas9技術(shù)構(gòu)建可誘導(dǎo)表達(dá)基因編輯慢病毒載體系統(tǒng),并且在MKN-45細(xì)胞系驗證其基因編輯功能。方法:分別以lenti-cas9-BLAST和PX458質(zhì)粒為模板,擴(kuò)增Blasticidin s及T2A-GFP基因片段,凝膠電泳回收后與Lenti-guide-puro質(zhì)粒重組,獲得重組質(zhì)粒Lenti-guide-BLAST-GFP。針對CXCR4基因EXON2設(shè)計并合成三條sg RNA,與Lenti-guide-BLAST-GFP重組獲得Lenti-guide-BLAST-GFP-sgRNA質(zhì)粒,擴(kuò)增后進(jìn)行慢病毒載體包裝。對pCW-Cas9質(zhì)粒擴(kuò)增后進(jìn)行慢病毒包裝。然后將上述兩種載體依次感染MKN45細(xì)胞,壓力篩選培養(yǎng),經(jīng)DOX 1μg/ml誘導(dǎo)后,檢測MKN-45細(xì)胞CXCR4蛋白表達(dá)和基因突變水平,并驗證CXCR4基因編輯前后MKN-45細(xì)胞對SDF-1應(yīng)答改變。結(jié)果:經(jīng)過測序證實,成功構(gòu)建了Lenti-guide-BLAST-GFP和Lenti-guide-BLAST-GFP-sgRNA質(zhì)粒,并成功獲得Lenti-guide-BLAST... 

【文章來源】：重慶醫(yī)科大學(xué)重慶市

【文章頁數(shù)】：43 頁

【學(xué)位級別】：碩士

【部分圖文】：

三條gRNA成功克隆至重真核表質(zhì)粒Lenti-guide-BLAST-GFPFigure1ResultsofSangersequencingconfirmingthatthethreegRNAswereinsertedintothelenti-guide-BLAST-GFPplasmid.

圖 2 Western Blot 檢測 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 .包裝 GFP 標(biāo)記的 gRNA 慢病毒載體感染慢病毒載體后，經(jīng)歷再次抗菌素壓力篩選，熒光顯微鏡鏡檢胞表達(dá) GFP，陽性率在 90%以上（圖 3）。

圖 2 Western Blot 檢測 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 成功包裝 GFP 標(biāo)記的 gRNA 慢病毒載體二次感染慢病毒載體后，經(jīng)歷再次抗菌素壓力篩選，熒光顯微鏡鏡檢 MKN-45細(xì)胞，細(xì)胞表達(dá) GFP，陽性率在 90%以上（圖 3）。

【參考文獻(xiàn)】：
期刊論文
[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)



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