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一种基于报告基因的细胞检测,用于监测剪接效率
JoVE Journal
Biochimica
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JoVE Journal Biochimica
A Reporter Based Cellular Assay for Monitoring Splicing Efficiency
DOI:

08:53 min

September 15, 2021

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Capitoli

  • 00:04Introduction
  • 00:31Cotransfection of HeLa Cells with the Reporter and U1 Small Nuclear RNA (snRNA) Plasmids
  • 02:0032P-Labeling of Oligonucleotides
  • 02:52Analysis of the Spliced Reporter Transcripts by Primer Extension
  • 03:53Analysis of the Spliced Reporter Transcripts by Fluorescent RT-PCR
  • 05:08Analysis of Variant U1 snRNA Expression by RT-qPCR
  • 05:38Setup and Running of Urea-PAGE Gels
  • 06:27Results: Reporter Based Splicing Efficiency of U1 snRNA Constructs
  • 08:09Conclusion

Summary

Traduzione automatica

该协议描述了一种微型基因报告基因检测,用于监测5'-剪接位点突变对剪接的影响,并开发抑制因子U1 snRNA以挽救突变诱导的剪接抑制。报告基因和抑制子U1 snRNA构建体在HeLa细胞中表达,并通过引物延伸或RT-PCR分析剪接。

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