| 郗永义,吴晓洁,林艳丽,钟荣斌,周艳荣,陈红星,王友亮.基于CRISPR/Cas9基因编辑技术的EGFP基因定点整合表达[J].现代生物医学进展英文版,2018,(19):3601-3606. |
| 基于CRISPR/Cas9基因编辑技术的EGFP基因定点整合表达 |
| Site-directed Integrated Expression of EGFP Gene Based on CRISPR / Cas9 Gene Editing |
| Received:March 28, 2018 Revised:April 25, 2018 |
| DOI：10.13241/j.cnki.pmb.2018.19.001 |
| 中文关键词: 基因编辑 CRISPR/Cas9 同源置换 定点整合 |
| 英文关键词: Gene editing CRISPR/Cas9 Homologous substitution Site-specific integration |
| 基金项目:国家重点实验室基金项目（SKLPBS1444）；全军医学科技研究基金项目（16QNP126） |
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| 中文摘要: |
| 摘要 目的：利用CRISPR/Cas9基因编辑技术，实现EGFP基因在CHO细胞ACTB基因座位置定点整合和表达，建立基于CRISPR/Cas9技术的外源基因定点整合和表达技术。方法：根据CHO细胞β-actin(ACTB)基因起始密码子区基因序列，设计相应CRISPR/Cas9系统，同时构建含有ACTB同源臂和EGFP基因的同源供体载体（donor vector），通过脂质体转染法同时转染CRISPR/Cas9和供体载体，流式分选EGFP阳性细胞，分析基因编辑技术在EGFP基因定点整合和表达方面的可行性。结果：构建了能有效切割CHO细胞ACTB基因的CRISPR/Cas9系统，筛选到EGFP定点整合至ACTB基因座并有效表达的细胞，ACTB基因缺失后由于γ-actin代偿性表达增强，ACTB缺失细胞形态和生长未受影响。结论：单纯依靠基因编辑技术可以实现1 kb以内的基因同源置换，但效率较低，如实现更大片段的外源基因置换，需借助其它实验技术。 |
| 英文摘要: |
| ABSTRACT Objective: To use CRISPR / Cas9 gene editing technology to achieve the site-specific integration and expression of EGFP gene in the ACTB locus of CHO cells, and establish the site-directed integration and expression of exogenous genes based on CRISPR / Cas9 technology. Methods: The CRISPR / Cas9 system was designed according to the start codon region of β-actin (ACTB) gene in CHO cells. At the same time, a homologous donor vector containing ACTB homology arm and EGFP gene was constructed. The transfection of CRISPR / Cas9 plasmid and donor vector was carried out by Liposomes transfection method. The EGFP positive cells were sorted by flow cytometry. The feasibility of gene editing in the site-specific integration and expression of EGFP gene was analyzed. Results: The CRISPR / Cas9 system effectively cut the ACTB gene of CHO cells. The EGFP expressed efficiently in the ACTB locus. After ACTB gene deletion, the compensatory expression of γ-actin was enhanced, and CHO cell growth well. Conclusion: Genomic re- placement within 1 kb can be achieved by simply relying on gene editing techniques, but the efficiency is low. To achieve exogenous gene replacement of larger fragments, other experimental techniques are needed. |
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