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Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes
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Bioengenharia
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JoVE Journal Bioengenharia
Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes

Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes

DOI:
10.3791/63828-v

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09:45 min

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June 02, 2022

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, , , ,
1Department of Bioengineering,Clemson University, 2Godley Snell Research Center,Clemson University

Capítulos

  • 00:04Introduction
  • 01:18Animal Surgery
  • 03:26Hepatocyte Isolation
  • 04:54Electroporation and Cell Culture
  • 07:46Results: Generating Electroporation-Assisted CRISPR-Cas9 Edits in Freshly Isolated Mouse Hepatocytes
  • 09:06Conclusion

Summary

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This protocol describes techniques for isolating primary mouse hepatocytes from the liver and electroporating CRISPR-Cas9 as ribonucleoproteins and mRNA to disrupt a therapeutic target gene associated with an inherited metabolic disease of the liver. The methods described result in high viability and high levels of gene modification after electroporation.

Tags

Keywords: ElectroporationCas9 RibonucleoproteinsMRNAPrimary Mouse HepatocytesGene EditingLiver PerfusionHepatocyte IsolationIn Vitro And In Vivo Disease ModelsAnesthesiaPortal VeinInferior Vena CavaPerfusion SolutionFlow RateLiver SofteningCotton SwabForceps

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