Organoid Electroporation: A Method to Transfect Plasmid DNA into Gastrointestinal Organoids

Abstract

Source: Gaebler, A. M.et al.  Universal and Efficient Electroporation Protocol for Genetic Engineering of Gastrointestinal Organoids. J. Vis. Exp. (2020).

This video describes the electroporation protocol for transfecting large plasmids into three-dimensional gastrointestinal organoids to prove its universal functionality in different entities, namely pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), cholangiocarcinoma (CCC), and gastric cancer (GC) organoids.

Protocol

1. Organoid Culture and Preparations Before Electroporation Establish organoids by tissue digestion as described previously and expand them with their corresponding entity specific culture medium in a basement matrix overview see Table 1 and Table of Materials NOTE: For human tissue samples informed consent and approval of the study by an ethical committee is necessary. Prewarm 48-well plates at 37 °C for post-electroporation seeding.</l…

Representative Results

Figure 1: Electroporation preparation workflow. First, organoids ought to be dissociated to clusters of 10-15 cells and antibiotics should get washed out. After electroporation the white foam needs to be dissociated. Cells can be seeded after regenerating for 40 min at room temperature. P…

Materials

[Leu15] Gastrin	Sigma-Aldrich	G9145
A83-01	Tocris Bioscience	2939
Advanced DMEM/F-12	Invitrogen	12634010
B27	Invitrogen	17504044
B27 Supplement, minus vitamin A	Thermo Fisher Scientific	12587010
CHIR99021	Stemgent	04-2004
Collagenase II	Life Technologies	17101-015
Collagenase XI	Sigma-Aldrich	C9407-100MG
Collagenase D	Roche	11088866001
Dispase II	Roche	4942078001
Dnase I	Sigma-Aldrich	D5319
D-sorbitol	Roth	6213.1
Dithiothreitol	Thermo Scientific	1859330
EDTA	Roth	8040
Forskolin	Tocris Bioscience	1099
Glutamax	Life Technologies	35050061
Hepes	Thermo Fisher Scientific	15630106
hFGF-10	Preprotech	100-26
KCl	Sigma-Aldrich	P9541
KH2PO4	Roth	3904.2
Matrigel	Corning	356231	basement matrix
mEGF	Invitrogen	PMG8043
N2	Invitrogen	17502048
NaCl	Roth	3957.1
Na2HPO4	Roth	K300.2
N-Acetyl-L-Cystein	Sigma-Aldrich	A9165
Nicotinamid	Sigma-Aldrich	N0636
Noggin	n.a.	n.a.	Conditioned medium produced from HEK293 cells (Hek293- mNoggin-Fc)
Recombinant Human HGF	Preprotech	100-39H
Rspondin	n.a.	n.a.	Conditioned medium produced from HEK293 cells (HA-Rspo1- Fc-293T)
SB202190	Sigma-Aldrich	S7067
TrypLE Express	Gibco	12604021	Dissociation reagent
Wnt3A	n.a.	n.a.	Conditioned medium produced from L-Wnt3a cells (from Sylvia Boj)
Y-27632	Sigma-Aldrich	Y0503
48-well plate	Corning	3548
Nepa Electroporation Cuvettes 2mm gap w/pipettes	Nepa Gene Co., Ltd.	EC-002S
Electroporator Nepa21	Nepa Gene Co., Ltd.	n.a.
EVOS FL Auto	Invitrogen	AMAFD1000	Fluorescence microscope
EndoFree Plasmid Maxi Ki	Qiagen	12362
Opti-MEM	Gibco	31985047	Electroporation buffer
2 gRNA concatemer vector	AddGene	84879
px458 plasmid	AddGene	48138	coding for sgRNA and Cas9
px458_Conc2 plasmid	AddGene	134449	px458 plasmid containing 2x U6 promotors for two different sgRNAs