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

Published: April 30, 2023

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…

開示

The authors have nothing to disclose.

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

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記事を引用
Organoid Electroporation: A Method to Transfect Plasmid DNA into Gastrointestinal Organoids. J. Vis. Exp. (Pending Publication), e20305, doi: (2023).

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