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10:40 min
April 25th, 2022
DOI :
10.3791/63704-v
Chapters
0:04
Introduction
0:58
Preparation of CRISPR Gene Editing and Guide RNA Design to Generate miRNA Cluster Knockout Cell Lines
2:06
Generation of Stable Lentiviral Cell Lines Carrying the DOX-Inducible Cas9 Expression Cassette
3:20
Performing CRISPR Reactions by Cas9 Induction and Synthetic Guide RNA Transfection of Cells Using a High-Throughput Format
7:22
PCR Genotyping of CRISPR Cell Lines Using Crude Cell Lysates
8:37
Results: High-Throughput CRISPR Experimental Design to Generate Multiple miR-888 Cluster Deletion Combinations
9:51
Conclusion
Transcript
该协议使用高通量CRISPR基因编辑工作流程来分子剖析组织和聚集单位的micro RNA基因。并确定这些非编码RNA网络如何协调癌症进展途径。这种CRISPR基因编辑程序允许研究人员快速生成携带独特micro RNA簇缺失组合的整个细胞系组合,同时避免耗时的DNA载体亚克隆。
在将微RNA作为治疗和诊断工具翻译成临床之前,该方法将更清楚地了解簇状微RNA如何协同功能以调节肿瘤生长,侵袭性和耐药性。演示该程序的将是我实验室的研究助理Grace Yi。首先,使用 DNA 序列注释软
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Summary
该协议描述了用于microRNA聚类网络分析的高通量簇规则间隔短回文重复(CRISPR)基因编辑工作流程,允许在单个实验中快速生成一组携带独特miRNA簇成员缺失组合的转基因细胞系。
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