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Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System
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JoVE Journal Medicina
Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System

DOI:
10.3791/61987-v

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07:04 min

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February 04, 2021

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, , , , , , ,
1Department of Ophthalmology,University Hospital RWTH Aachen, 2Experimental Ophthalmology,University of Geneva, 3Department of Ophthalmology,University Hospitals of Geneva, 4Université de Paris, CNRS, INSERM, UTCBS, Unité des technologies Chimiques et Biologiques pour la Santé, 5Chimie ParisTech,PSL Research University, 6Max Delbrück Center for Molecular Medicine in the Helmholtz Association, 7Division of Medical Biotechnology,Paul-Ehrlich-Institute

Capitoli

  • 00:05Introduction
  • 00:57Primary Human RPE Cell Electroporation and Plasmid DNA Preparation
  • 01:57Cultivated Primary Human RPE Cell Preparation
  • 03:19His-Tagged Pigment Epithelium-Derived Factor (PEDF) Fusion Protein Purification
  • 04:57Results: Representative Primary Human RPE Transfection and Genetic Modification Evaluation
  • 06:24Conclusion

Summary

Traduzione automatica

Traduzione automatica

We have developed a protocol to transfect primary human pigment epithelial cells by electroporation with the gene encoding pigment epithelium-derived factor (PEDF) using the Sleeping Beauty (SB) transposon system. Successful transfection was demonstrated by quantitative polymerase chain reaction (qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA).

Tags

Keywords: ElectroporationSleeping Beauty Transposon SystemPrimary Human Pigment Epithelial CellsTransgene ExpressionProtein SecretionRetinal Degenerative DiseasesGene Addition TherapyPlasmid DNATransposasePigment Epithelium Derived FactorBuffer ETrypsin EDTAPBSBuffer R

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