Universal and Efficient Electroporation Protocol for Genetic Engineering of Gastrointestinal Organoids

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.

Analyzing efficiency of a lentiviral shRNA knockdown system in human enteroids using western blot and flow cytometry

“… 1 Enteroids provide a highly translational model of the human intestine. 2 , 3 , 4 While various protocols have been described to genetically modify enteroids, 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 technical challenges have often limited efficacy of genetic manipulation in these cultures. 14 , 15 , 16 Here, we introduce a procedure predicated upon methodology from Maru et al.…”

The Power of Gene Technologies: 1001 Ways to Create a Cell Model

“…A significant limitation of such 3D structures is their relatively small size (thickness) and the restricted number of layers, which is due to the absence of capillaries and adequate trophism in the thickness of such structures. The genetic modification of whole organoids using electroporation and viral particles is also possible, although the multilayer nature of an organoid greatly reduces its effectiveness for studying cells in the inner layers [ 136 , 137 ]. Moreover, genetic modification of the whole organoid may cause genetic and phenotypic heterogeneity between its cells, that decreases the quality and reproducibility of the results obtained.…”