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整合酶缺陷慢病毒载体载体的制备及其在细胞 CRISPR/Cas9-mediated 基因敲除中的表达
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JoVE Journal Genética
A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells
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整合酶缺陷慢病毒载体载体的制备及其在细胞 CRISPR/Cas9-mediated 基因敲除中的表达

DOI:
10.3791/56915-v

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10:42 min

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December 12, 2017

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1Duke Viral Vector Core, Department of Neurobiology,Duke University School of Medicine

Capítulos

  • 00:05Título
  • 00:52Transfecting of HEK-293T Cells Using Calcium Phosphate
  • 02:19Harvesting Virus
  • 02:50Concentration of Viral Particles by Ultracentrifugation
  • 05:11Esimating of Viral Titers Using p24-enzyme-linked Immunosorbent Assay
  • 07:31Counting GFP-positive Cells
  • 08:25Results: Production and Validation of the Knockout-efficiency of Integrase-deficient Lentiviral-CRSPER/Cas9 Vector
  • 09:57Conclusion

Summary

Tadução automática

Tadução automática

我们描述了整合酶缺乏慢病毒载体载体 (IDLVs) 作为向细胞传递 CRISPR/Cas9 的工具的生产策略。由于能够在细胞中调节快速和健壮的基因编辑, IDLVs 为基因的传递提供了一个更安全、同样有效的载体平台, 与整合酶能力的载体相比。

Tags

Integrase-deficient Lentiviral VectorsCRISPR/Cas9Gene EditingHEK-293T CellsTransfectionSucrose GradientUltracentrifugation

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