Universal and Efficient Electroporation Protocol for Genetic Engineering of Gastrointestinal Organoids

J Vis Exp. 2020 Feb 18;(156). doi: 10.3791/60704.

Abstract

Electroporation is a common method for transfection with different kinds of molecules by electrical permeabilization of the plasma membrane. With the increasing use of organoids as a culturing method for primary patient material in the last years, efficient transfer methods of components for genetic engineering in this 3D culture system are in need. Especially for organoids, the efficiency of genetic manipulations depends on a successful transfection. Thus, this protocol was developed to facilitate the electroporation of organoids and to prove its universal functionality in different entities. Human colorectal, pancreatic, hepatic and gastric cancer organoids were successfully electroporated with small and large plasmids in comparison. Based on GFP encoding vectors, the transfection efficiency was determined by FACS. No extensive preparation of the cells or special, cost-intensive electroporation buffers are necessary, and the protocol can be performed within one day.

Publication types

  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Colorectal Neoplasms / pathology*
  • Electroporation / methods*
  • Genetic Engineering / methods*
  • Humans
  • Liver Neoplasms / pathology*
  • Organ Culture Techniques
  • Organoids / pathology*
  • Pancreatic Neoplasms / pathology*
  • Plasmids / administration & dosage*
  • Plasmids / genetics
  • Stomach Neoplasms / pathology*
  • Transfection