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A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells
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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

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

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

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We describe the production strategy of integrase-deficient lentiviral vectors (IDLVs) as vehicles for delivering CRISPR/Cas9 to cells. With an ability to mediate quick and robust gene editing in cells, IDLVs present a safer and equally effective vector platform for gene delivery compared to integrase-competent vectors.

Tags

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

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