We thank Juliane Fohgrub, Ann-Christin Meinecke and Max Heiduk for excellent technical assistance. Funding was provided by Deutsche Krebshilfe (No 111350 and 70112925), Sander Stiftung (No 2014.104.1), Hector Stiftung (No M65.2) and the European Union (ERC No 639050).

Ethics approval was obtained from the ethical committee of the TU Dresden (#EK451122014).
1. Organoid Culture and Preparations Before Electroporation
<ol>
	<li>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)11,12,13,14,15,16,17. 
	NOTE: For human tissue samples informed consent and approval of the study by an ethical committee is necessary.</li>
	<li>Prewarm 48-well plates at 37 &#176;C for post-electroporation seeding.</li>
	<li>Prepare basal medium w/o antibiotics as well as entity specific organoid culture medium w/o antibiotics (see Table 1) including 10 &#181;M Y-27632 and 3 &#181;M CHIR99021. 
	NOTE: The withdrawal of antibiotics is important to reduce toxic effects. Y-27632 and CHIR99021 ameliorate cell recovery10.</li>
	<li>Preparation of the organoids (see Figure 1)
	<ol>
		<li>Cultivate 5 wells of organoids in a 48-well plate per electroporation sample in culture medium. 
		NOTE: Proliferative organoids should be used (around 2-3 days after last splitting).</li>
		<li>Prepare 230 &#181;L of dissociation reagent (see Table of Materials) including 10 &#181;M Y-27632 per well.</li>
		<li>Remove the culture medium from each well and dissociate the organoids mechanically in 230 &#181;L of the prepared dissociation mixture. Pool 5 wells per electroporation sample into one 15 mL tube.</li>
		<li>Mix by vortexing and incubate for 5-15 min at 37 &#176;C until clusters of 10-15 cells occur. Therefore, check the dissociation microscopically. Stop the digestion by adding basal medium w/o antibiotics up to 10 mL. 
		NOTE: This step is very critical! The electroporation efficiency will be reduced, when incubation is too short, but long digestion will reduce survivability.</li>
		<li>Centrifuge at 450 x g for 5 min at room temperature, discard the supernatant and wash twice with 4 mL of electroporation buffer (see Table of Materials).</li>
	</ol>
	</li>
</ol>
2. Electroporation
NOTE: The following protocol is developed for electroporators capable of square waves and separated poring and transfer pulse sequences (see Figure 2). Optionally, impedance values as well as the voltages, currents and energies transferred into the sample can be measured as control for reproducible experiments.
<ol>
	<li>Resuspend the organoid pellet in 100 &#181;L of electroporation buffer (see Table of Materials) containing 30 &#181;g of plasmid DNA. 
	NOTE: The concentration of the used plasmid DNA should exceed 5 &#181;g/&#181;L for an optimal salt concentration during the electroporation process. Therefore, endofree plasmid maxi kits (see Table of Materials) for the preparation of vectors are recommended. A total amount up to 45 &#181;g of DNA can be used without cytotoxic effects.</li>
	<li>Dispense the complete DNA-organoid mixture into an electroporation cuvette with 2 mm gap width without producing air bubbles.</li>
	<li>Set the electroporation parameters according to Fujii et al.10 (see Table 2, Figure 2).</li>
	<li>Mix the cells slightly without foaming by tapping the cuvette with a finger. Place the cuvette into the cuvette chamber.</li>
	<li>Press the &#937; button of the electroporator and make a note of the impedance value. 
	NOTE: An impedance between 30-40 &#937; showed the best results. In general, it should be in the range between 30-55 &#937;. If this is not the case, please control the following aspects: gap width of the cuvette used, cable connections of the electroporator, possible air bubbles, correct volume and salt concentration of the electroporation mixture.</li>
	<li>Press the Start button to execute the electroporation program and control the values of currents, voltages and energies displayed. 
	NOTE: The values of measured voltages, currents and energies should correspond to the set electroporation parameters. For the comparison of repeated experiments, it can be helpful to note these data.</li>
	<li>After electroporation, immediately add 500 &#181;L of culture medium w/o antibiotics (with CHIR99021 and Y-27632; see step 1.3). Mix by pipetting up and down to dissociate the white foam. 
	NOTE: The white foam appears after the electroporation process and a significant number of cells is attached to it. So, dissociation of it is very important for not losing cells.</li>
	<li>Transfer the sample completely from the cuvette into a new 15 mL tube using the pipette belonging to the electroporation cuvettes (see Table of Materials). Rinsing the cuvette again with basal medium is recommended to obtain remaining cells.</li>
	<li>For regeneration of the cells, incubate them for 40 min at room temperature.</li>
</ol>
3. Seeding of Cells
<ol>
	<li>Centrifuge the cells at 450 x g for 5 min at room temperature and discard the supernatant.</li>
	<li>Resuspend the pellet in 100 &#181;L of basement matrix and seed 20 &#181;L drops in a prewarmed 48-well plate (see step 1.2). Incubate for 10 min at 37 &#176;C for polymerization and add 250 &#181;L of culture medium, which is supplemented with Y-27632 and CHIR99021 until the next splitting of the grown organoids (around 5-7 days).</li>
</ol>
4. Determination of Transfection Efficiency
NOTE: In general, it is recommended to electroporate a vector carrying a fluorescence marker as additional transfection control. Dependent on the chosen marker and its chromophore maturation the fluorescence will be visible within around 24-48 h post transfection18.
<ol>
	<li>Check the fluorescence microscopically after 24-48 h in the transfection control (see Figure 4B).</li>
	<li>Fluorescent activated cell scanning (FACS)
	<ol>
		<li>Harvest the cells analogously to step 1.4.2-1.4.4 and digest around 10-20 min until there are single cells. Add up to 10 mL of phosphate-buffered saline (PBS).</li>
		<li>Centrifuge at 450 x g for 5 min at room temperature and discard the supernatant.</li>
		<li>Optionally for discrimination of living cells: Resuspend the pellet in 1 mL of phosphate-buffered saline (PBS) and add a suitable antibody (see Table of Materials) or propidium iodide (PI). Mix very carefully only by tapping and incubate for 30 min at room temperature in the dark. Wash with 10 mL of PBS, centrifuge and discard the supernatant.</li>
		<li>Resuspend the cell pellet in 200 &#181;L of PBS and optionally filter the suspension through a 100 &#181;m cell strainer into a FACS tube.</li>
		<li>Analyze the cells by a FACS machine using an appropriate gating strategy (see Figure 3; Figure 4A) and determine the transfection efficiency.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Huch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+culture+of+genome-stable+bipotent+stem+cells+from+adult+human+liver.">Long-term culture of genome-stable bipotent stem cells from adult human liver.</a> Cell. 160 (1-2), 299-312 (2015).</li><li>Werner, K., Weitz, J., Stange, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+as+Model+Systems+for+Gastrointestinal+Diseases:+Tissue+Engineering+Meets+Genetic+Engineering.">Organoids as Model Systems for Gastrointestinal Diseases: Tissue Engineering Meets Genetic Engineering.</a> Current Pathobiology Reports. 4 (1), 1-9 (2016).</li><li>Olayanju, A., Jones, L., Greco, K., Goldring, C. E., Ansari, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+porcine+gastrointestinal+organoid+units+as+a+potential+in+vitro+tool+for+drug+discovery+and+development.">Application of porcine gastrointestinal organoid units as a potential in vitro tool for drug discovery and development.</a> Journal of applied toxicology. 39 (1), 4-15 (2019).</li><li>Drost, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+cancer+mutations+in+cultured+human+intestinal+stem+cells.">Sequential cancer mutations in cultured human intestinal stem cells.</a> Nature. 521 (7550), 43-47 (2015).</li><li>Matano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+colorectal+cancer+using+CRISPR-Cas9-mediated+engineering+of+human+intestinal+organoids.">Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids.</a> Nature Medicine. 21 (3), 256-262 (2015).</li><li>Schwank, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+repair+of+CFTR+by+CRISPR/Cas9+in+intestinal+stem+cell+organoids+of+cystic+fibrosis+patients.">Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients.</a> Cell Stem Cell. 13 (6), 653-658 (2013).</li><li>Drost, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+CRISPR-modified+human+stem+cell+organoids+to+study+the+origin+of+mutational+signatures+in+cancer.">Use of CRISPR-modified human stem cell organoids to study the origin of mutational signatures in cancer.</a> Science. 358 (6360), 234-238 (2017).</li><li>Zhang, X. H., Tee, L. Y., Wang, X. G., Huang, Q. S., Yang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Off-target+Effects+in+CRISPR/Cas9-mediated+Genome+Engineering.">Off-target Effects in CRISPR/Cas9-mediated Genome Engineering.</a> Molecular Therapy Nucleic Acids. 4, e264 (2015).</li><li>Tsong, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+cell+membranes.">Electroporation of cell membranes.</a> Biophysical journal. 60 (2), 297-306 (1991).</li><li>Fujii, M., Matano, M., Nanki, K., Sato, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+genetic+engineering+of+human+intestinal+organoids+using+electroporation.">Efficient genetic engineering of human intestinal organoids using electroporation.</a> Nature Protocols. 10 (10), 1474-1485 (2015).</li><li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+expansion+of+epithelial+organoids+from+human+colon,+adenoma,+adenocarcinoma,+and+Barrett's+epithelium.">Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.</a> Gastroenterology. 141 (5), 1762-1772 (2011).</li><li>Bartfeld, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+expansion+of+human+gastric+epithelial+stem+cells+and+their+responses+to+bacterial+infection.">In vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection.</a> Gastroenterology. 148 (1), 126-136 (2015).</li><li>Boj, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+models+of+human+and+mouse+ductal+pancreatic+cancer.">Organoid models of human and mouse ductal pancreatic cancer.</a> Cell. 160 (1-2), 324-338 (2015).</li><li>Broutier, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+and+establishment+of+self-renewing+human+and+mouse+adult+liver+and+pancreas+3D+organoids+and+their+genetic+manipulation.">Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation.</a> Nature Protocols. 11 (9), 1724-1743 (2016).</li><li>Broutier, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+primary+liver+cancer-derived+organoid+cultures+for+disease+modeling+and+drug+screening.">Human primary liver cancer-derived organoid cultures for disease modeling and drug screening.</a> Nature Medicine. 23 (12), 1424-1435 (2017).</li><li>Hennig, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CFTR+Expression+Analysis+for+Subtyping+of+Human+Pancreatic+Cancer+Organoids.">CFTR Expression Analysis for Subtyping of Human Pancreatic Cancer Organoids.</a> Stem Cells Int. 2019, 1024614 (2019).</li><li>Seidlitz, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+gastric+cancer+modelling+using+organoids.">Human gastric cancer modelling using organoids.</a> Gut. 68 (2), 207-217 (2019).</li><li>Stepanenko, O. V., Verkhusha, V. V., Kuznetsova, I. M., Uversky, V. N., Turoverov, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+proteins+as+biomarkers+and+biosensors:+throwing+color+lights+on+molecular+and+cellular+processes.">Fluorescent proteins as biomarkers and biosensors: throwing color lights on molecular and cellular processes.</a> Current protein &amp; peptide science. 9 (4), 338-369 (2008).</li><li>Andersson-Rolf, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+paralogue+knockout+using+a+CRISPR-concatemer+in+mouse+small+intestinal+organoids.">Simultaneous paralogue knockout using a CRISPR-concatemer in mouse small intestinal organoids.</a> Dev Biol. 420 (2), 271-277 (2016).</li><li>Ran, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nature Protocols. 8, 2281 (2013).</li><li>Merenda, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Protocol+for+Multiple+Gene+Knockout+in+Mouse+Small+Intestinal+Organoids+Using+a+CRISPR-concatemer.">A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer.</a> Journal of Visualized Experiments. (125), (2017).</li><li>Lesueur, L. L., Mir, L. M., Andre, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overcoming+the+Specific+Toxicity+of+Large+Plasmids+Electrotransfer+in+Primary+Cells+In+Vitro.">Overcoming the Specific Toxicity of Large Plasmids Electrotransfer in Primary Cells In Vitro.</a> Molecular Therapy Nucleic Acids. 5, e291 (2016).</li><li>Watanabe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nature Biotechnology. 25 (6), 681-686 (2007).</li><li>Darquet, A. M., Cameron, B., Wils, P., Scherman, D., Crouzet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+DNA+vehicle+for+nonviral+gene+delivery:+supercoiled+minicircle.">A new DNA vehicle for nonviral gene delivery: supercoiled minicircle.</a> Gene Therapy. 4 (12), 1341-1349 (1997).</li><li>Jacobi, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+CRISPR+tools+for+efficient+genome+editing+and+streamlined+protocols+for+their+delivery+into+mammalian+cells+and+mouse+zygotes.">Simplified CRISPR tools for efficient genome editing and streamlined protocols for their delivery into mammalian cells and mouse zygotes.</a> Methods. 121-122, 16-28 (2017).</li></ol>

The authors have nothing to disclose.

This protocol gives detailed instructions for an efficient, quick and easy to perform electroporation of different organoid entities. Additional to the presented tumor organoids from PDAC, CRC, CCC and GC, it works successfully for organoids derived from healthy tissue as well. The protocol can be performed within one day. In published organoid transfection protocols the whole procedure lasted four days including two days of preparations with different types of cultivation media10,21. In our protocol no special pretreatment is required. By washing with electroporation buffer before electroporation the antibiotic components of the media were washed out and an adjustment of saline concentrations for optimal impedance values was achieved. Nevertheless, some critical aspects should be considered for a successful electroporation:
Cells 
In the electroporation protocol by Fujii et al.10 it is recommended to dissociate organoids to single cells and to filter them through a 20 &#181;m cell-strainer. In our hands digestion to single cells strongly decreases the survivability of cells. As suggested in Merenda et al.21, we also dissociated organoids to clusters of 10-15 cells and could not determine a decreased efficiency compared to single cell dissociation. After electroporation, it is a very important step to dissociate the white foam, so that no attached cells are getting lost.
For 2D cell culture, it has been shown that a regeneration time after electroporation of more than 10 min up to 40 min increases survivability and transfection efficiency especially of large plasmids22. In test experiments, the same could be documented for organoids, leading to an incubation step of 40 min after electroporation in this protocol. In order to increase recovery from the electroporation, we cultured them with Rho-associated protein kinase (ROCK) inhibitor Y-27632 for five to seven days23. Similarly, the additional supplementation of glycogen synthase kinase 3 (GSK3) inhibitor CHIR99021 is meant to help single cells to recover10.
Settings 
One of the advantages of the used electroporator is that the impedance can be measured before electroporation for optimal conditions. According to the manufacturer, the impedance values should be 30-55 &#937;. In our hands, impedance values of 30-40 &#937; have shown optimal efficiencies. In a preliminary experiment, different voltages and pulse length values of the poring pulse were varied to find the optimal proportion of efficiency to survivability. In summary, we could confirm the described values of Fujii et al.10 in the different entities described here.
DNA 
The effect of different DNA amounts was tested in preliminary experiments up to 45 &#181;g of DNA per sample. No cytotoxic effects could be detected. Transfection efficiency was increased in a dose dependent way with saturation &#62; 30 &#181;g. So, we used 30 &#181;g per sample in the final protocol, but of course it can be increased (e.g., for the electroporation of more plasmids in parallel). Additionally, the purity and concentration of the DNA seems to be very important. A concentration exceeding 5 &#181;g/&#181;L has shown optimal transfection efficiencies.
As expected, the 9.3 kB plasmid could be transfected with a lower efficiency than the smaller 4.2 kB plasmid (see Figure 4). The use of even larger plasmids than 10 kB is anticipated to further decrease the efficiency. For future applications, it might be interesting to test minicircle DNA as a vector, since these gene carriers lack the bacterial backbone of a plasmid which makes them smaller24. This should result in an enhanced transfection efficiency. Furthermore, for CRISPR-based manipulations of organoids a direct electroporation of sgRNAs bound to Cas9 as a ribonucleoprotein (RNP) complex could be an alternative or addition25.

In recent years, a novel 3D cell culture system, termed organoids, was developed for various normal and cancerous tissues. Organoids are functionally and morphologically very close to their tissue of origin. They can be generated from different species, are easily expandable, genomically stable and genetically modifiable, which makes them an ideal model system for genetic investigations1,2,3. Genetic engineering techniques like the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system enable diverse manipulations. The selection of clones can be realized by defined media conditions, for example, by WNT ligand withdrawal for APC (Adenomatosis Polyposis Coli) knockout clones4,5. Alternatively, selection markers have to be introduced by homologous directed repair of a targeting vector6,7. Due to the fact that often more than one plasmid needs to be introduced, an efficient transfection becomes a crucial parameter. Additionally, to reduce unspecific off-target effects, a transient expression of the Cas9 endonuclease is desirable8.
Electroporation is a comparatively simple method to transfect cells with DNA, RNA, proteins or other macromolecules. By means of electric pulses, the cell membrane becomes more permeable and causes an increased uptake9. In a previously published electroporation protocol of colon organoids a 30 % efficiency with a piggy-bac GFP (green fluorescent protein) expressing vector (7.4 kB) was reached in a four days procedure10. The following protocol was developed to facilitate an efficient transfection of cancerous or healthy organoids with large plasmids encoding for single guide RNA (sgRNA) and the Cas9 endonuclease sequence (e.g. px458 as vector with 9.3 kb). The whole electroporation process can be performed within one day, without special electroporation buffers, and with at least comparable efficiencies between different gastrointestinal organoids, namely pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), cholangiocarcinoma (CCC) and gastric cancer (GC) organoids.

<table><tbody><tr><td>&lt;strong&gt;For establishment and culture medium&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>[Leu15] Gastrin</td><td>Sigma-Aldrich</td><td>G9145</td><td></td></tr><tr><td>A83-01</td><td>Tocris Bioscience</td><td>2939</td><td></td></tr><tr><td>Advanced DMEM/F-12</td><td>Invitrogen</td><td>12634010</td><td></td></tr><tr><td>B27</td><td>Invitrogen</td><td>17504044</td><td></td></tr><tr><td>B27 Supplement, minus vitamin A</td><td>Thermo Fisher Scientific</td><td>12587010</td><td></td></tr><tr><td>CHIR99021</td><td>Stemgent</td><td>04-0004</td><td></td></tr><tr><td>Collagenase II</td><td>Life Technologies</td><td>17101-015</td><td></td></tr><tr><td>Collagenase XI</td><td>Sigma-Aldrich</td><td>C9407-100MG</td><td></td></tr><tr><td>Collagenase D</td><td>Roche</td><td>11088866001</td><td></td></tr><tr><td>Dispase II</td><td>Roche</td><td>4942078001</td><td></td></tr><tr><td>Dnase I</td><td>Sigma-Aldrich</td><td>D5319</td><td></td></tr><tr><td>D-sorbitol</td><td>Roth</td><td>6213.1</td><td></td></tr><tr><td>Dithiothreitol</td><td>Thermo Scientific</td><td>1859330</td><td></td></tr><tr><td>EDTA</td><td>Roth</td><td>8040</td><td></td></tr><tr><td>Forskolin</td><td>Tocris Bioscience</td><td>1099</td><td></td></tr><tr><td>Glutamax</td><td>Life Technologies</td><td>35050061</td><td></td></tr><tr><td>Hepes</td><td>Thermo Fisher Scientific</td><td>15630106</td><td></td></tr><tr><td>hFGF-10</td><td>Preprotech</td><td>100-26</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9541</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Roth</td><td>3904.2</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>356231</td><td>basement matrix</td></tr><tr><td>mEGF</td><td>Invitrogen</td><td>PMG8043</td><td></td></tr><tr><td>N2</td><td>Invitrogen</td><td>17502048</td><td></td></tr><tr><td>NaCl</td><td>Roth</td><td>3957.1</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Roth</td><td>K300.2</td><td></td></tr><tr><td>N-Acetyl-L-Cystein</td><td>Sigma-Aldrich</td><td>A9165</td><td></td></tr><tr><td>Nicotinamid</td><td>Sigma-Aldrich</td><td>N0636</td><td></td></tr><tr><td>Noggin</td><td>n.a.</td><td>n.a.</td><td>Conditioned medium produced from HEK293 cells (Hek293-mNoggin-Fc)</td></tr><tr><td>PBS</td><td>Gibco</td><td>14190169</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>Primocin</td><td>InvivoGen</td><td>ant-pm-1</td><td></td></tr><tr><td>Prostaglandin E2</td><td>Tocris Bioscience</td><td>2296</td><td></td></tr><tr><td>Recombinant Human HGF</td><td>Preprotech</td><td>100-39H</td><td></td></tr><tr><td>Rspondin</td><td>n.a.</td><td>n.a.</td><td>Conditioned medium produced from HEK293 cells (HA-Rspo1-Fc-293T)</td></tr><tr><td>SB202190</td><td>Sigma-Aldrich</td><td>S7067</td><td></td></tr><tr><td>Sucrose</td><td>VWR</td><td>27,480,294</td><td></td></tr><tr><td>TrypLE Express</td><td>Gibco</td><td>12604021</td><td>Dissociation reagent</td></tr><tr><td>Wnt3A</td><td>n.a.</td><td>n.a.</td><td>Conditioned medium produced from L-Wnt3a cells (from Sylvia Boj)</td></tr><tr><td>Y-27632</td><td>Sigma-Aldrich</td><td>Y0503</td><td></td></tr><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cell Strainer 100 &amp;micro;m</td><td>Falcon</td><td>352360</td><td></td></tr><tr><td>48-well plate</td><td>Corning</td><td>3548</td><td></td></tr><tr><td>Nepa Electroporation Cuvettes 2mm gap w/pipettes</td><td>Nepa Gene Co., Ltd.</td><td>EC-002S</td><td></td></tr><tr><td>Tubes 15 ml</td><td>Greiner</td><td>188271</td><td></td></tr><tr><td>Tubes 15 ml low binding</td><td>Eppendorf</td><td>30122208</td><td>Tubes for FACS preparing</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Electroporator Nepa21</td><td>Nepa Gene Co., Ltd.</td><td>n.a.</td><td></td></tr><tr><td>EVOS FL Auto</td><td>Invitrogen</td><td>AMAFD1000</td><td>Fluorescence microscope</td></tr><tr><td>LSRFortessa</td><td>BD Bioscience</td><td>647800E6</td><td>FACS</td></tr><tr><td>&lt;strong&gt;Reagents and plasmids&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Live/dead fixable blue dead cell stain kit</td><td>Invitrogen</td><td>L34962</td><td>Includes antibody for live/dead staining for FACS analysis</td></tr><tr><td>EndoFree Plasmid Maxi Kit</td><td>Qiagen</td><td>12362</td><td></td></tr><tr><td>Nutlin-3</td><td>Selleckchem</td><td>S1061/07</td><td></td></tr><tr><td>Opti-MEM</td><td>Gibco</td><td>31985047</td><td>Electroporation buffer</td></tr><tr><td>2 gRNA concatemer vector</td><td>AddGene</td><td>84879</td><td></td></tr><tr><td>pCMV-EGFP</td><td>Nepa Gene Co., Ltd.</td><td>n.a.</td><td></td></tr><tr><td>px458 plasmid</td><td>AddGene</td><td>48138</td><td>coding for sgRNA and Cas9</td></tr><tr><td>px458_Conc2 plasmid</td><td>AddGene</td><td>134449</td><td>px458 plasmid containing 2x U6 promotors for two different sgRNAs</td></tr><tr><td>sgRNA_hTP53_1a</td><td>Eurofins Genomics</td><td>n.a.</td><td>&lt;img src=&quot;/files/ftp_upload/60704/60704tomeq1.jpg&quot;/&gt;</td></tr><tr><td>sgRNA_hTP53_1b</td><td>Eurofins Genomics</td><td>n.a.</td><td>&lt;img src=&quot;/files/ftp_upload/60704/60704tomeq2.jpg&quot;/&gt;</td></tr><tr><td>sgRNA_hTP53_2a</td><td>Eurofins Genomics</td><td>n.a.</td><td>&lt;img src=&quot;/files/ftp_upload/60704/60704tomeq3.jpg&quot;/&gt;</td></tr><tr><td>sgRNA_hTP53_2b</td><td>Eurofins Genomics</td><td>n.a.</td><td>&lt;img src=&quot;/files/ftp_upload/60704/60704tomeq4.jpg&quot;/&gt;</td></tr></tbody></table>

universal and efficient electroporation protocol for genetic engineering of gastrointestinal organoids

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.

Organoids of four different cancer entities (CRC, CCC, PDAC, GC) were electroporated at least 3 times using 30 &#181;g of a small plasmid (pCMV-EGFP, 4.2 kb) or a large plasmid (px458, 9.3 kb). Both vectors carry a GFP cassette allowing the determination of transfection efficiency 48 h after electroporation by flow cytometry. To analyze only living cells, staining with a life-death antibody before scanning was performed. The gating strategy is shown in Figure 3.
In all four organoid entities the 4.2 kB sized plasmid was transfected with higher efficiency compared to the larger one (see Figure 4). The most efficient transfection of the small plasmid was reached in PDAC organoids with 92.1 &#177; 5.2 % GFP positive cells, whereas the large plasmid was transfected with an efficiency of 46.7 &#177; 3.7 % (mean &#177; standard deviation, n = 3). Compared to pancreatic cancer organoids, the larger plasmid was more efficiently transfected into CRC organoids with a mean efficiency of 53.4 &#177; 11.7 %, while the small plasmid was transfected with a mean efficiency of 84.3 &#177; 5.8 %. The most difficult entity to transfect were gastric cancer organoids: for both the large and the small plasmid, the lowest transfection efficiency was reached in this entity (32.3 &#177; 12.7 % and 74.1 &#177; 5.5 %, respectively). CCC organoids showed a mean transfection efficiency of 83.0 &#177; 13.1 % for the small plasmid and for the large plasmid 39.5 &#177; 10.4 % were obtained.
As proof of concept, human normal stomach organoids were electroporated with a px458_Conc2 plasmid encoding for Cas9, GFP and two sgRNAs targeting TP53. The Cas9-induced double strand breaks on exon 8 were repaired by non-homologous end-joining (NHEJ), resulting in frameshifts and consequently a knockout of the gene (see Supplementary Figure 1).
Table 1: Composition of basal media, digestion mixtures and cultivation media. <a href="https://www.jove.com/files/ftp_upload/60704/60704_table1.xlsx" target="_blank">Please click here to view this file (Right click to download).</a>
<img alt="Table 1" src="/files/ftp_upload/60704/60704table2.jpg" /> 
Table 2: Electroporation settings according to Fujii et al.10.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60704/60704fig1.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/60704/60704fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60704/60704fig2b.jpg" /> 
Figure 2: Two-step electroporation. Two poring pulses with higher voltage und short duration (175 V and 157.5 V, each for 5 ms, pause for 50 ms, voltage decay 10%) lead to the formation of pores in cell membranes. The following transfer pulses deliver the DNA into the cells: five positive transfer pulses (with 20 V, 12 V, 7.2 V, 4.32 V and 2,592 V, each for 50 ms, pause for 50 ms, voltage decay 40%), followed by five polarity exchanged transfer pulses (with 20 V, 12 V, 7.2 V, 4.32 V and 2,592 V, each for 50 ms, pause for 50 ms, voltage decay 40%). <a href="https://www.jove.com/files/ftp_upload/60704/60704fig2blarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60704/60704fig3b.jpg" /> 
Figure 3: Representative gating strategy shown by CCC organoids. All electroporated organoids were analyzed by flow cytometry 48 h after electroporation. Cells electroporated without plasmid DNA were used as negative controls. The gates were set as following: (A) gating for cell shape, (B,C) gating for single cells (doublet discrimination), (D) gating for living cells (stained with an antibody for apoptotic cells) and (E,F) finally gating for eGFP expressing cells (FITC channel). FSC = forward scatter; SSC = side scatter. <a href="https://www.jove.com/files/ftp_upload/60704/60704fig3blarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60704/60704fig4.jpg" /> 
Figure 4: Electroporation efficiency of four organoid entities. (A) FACS analysis (n = 34, mean standard deviation and each single value are shown) and (B) visual comparison by fluorescence microscope. Scale bar = 1,000 &#181;m. BF = bright field; CCC = cholangiocarcinoma; CRC = colorectal cancer; GC = gastric cancer; PDAC = pancreatic ductal adenocarcinoma. <a href="https://www.jove.com/files/ftp_upload/60704/60704fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplementary Figure 1" src="/files/ftp_upload/60704/60704suppfig1a.jpg" /> 
Supplementary Figure 1: Exemplary CRISPR/Cas9-based knockout of TP53 in normal human stomach organoids.The px458_Conc2 vector (see Table of Materials) was cloned by combining the 2 gRNA concatemer vector, a generous gift from Bon-Kyoung Koo19, with px45820, resulting in a plasmid encoding for 2 sgRNAs, Cas9 and GFP. Two sgRNAs targeting TP53 were introduced in px458_Conc2 vector by golden gate cloning (analogously to Andersson-Rolf et al.19). 10 &#181;g of plasmid DNA were electroporated in human normal gastric organoids (A). Clones were selected by Nutlin3 administration (B) and the TP53 knockout was confirmed by TOPO TA cloning and sequencing of the alleles, here exemplary shown for one clone (C). The sgRNAs are underlined in the reference. Scale bar = 200 &#181;m. BF = bright field. <a href="https://www.jove.com/files/ftp_upload/60704/60704suppfig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Universal and Efficient Electroporation Protocol for Genetic Engineering of Gastrointestinal Organoids at JoVE.com

Universal and Efficient Electroporation Protocol for Genetic Engineering of Gastrointestinal Organoids

This protocol describes an efficient electroporation method for the transfection of four different gastrointestinal organoid entities with larger plasmids (to the extent of 10 kB). It can be performed within one day and does not need extensive preparation or special, cost-intensive electroporation buffers.

Electroporation is a common method for transfection with different kinds of molecules by electrical permeabilization of the plasma membrane. With the ...

universal-efficient-electroporation-protocol-for-genetic-engineering

Research

JoVE Journal

Genetics

17.1K Views.  Technische Universität Dresden. This protocol describes an efficient electroporation method for the transfection of four different gastrointestinal organoid entities with larger plasmids (to the extent of 10 kB). It can be performed within one day and does not need extensive preparation or special, cost-intensive electroporation buffers.

Video: Universal and Efficient Electroporation Protocol for Genetic Engineering of Gastrointestinal Organoids

Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to manipulate gene expression in organoids have made limited progress in this arena. While CRISPR/Cas9 technology allows for precise genome editing of cells for organoid generation, this strategy requires extensive selection and screening by sequence analysis, which is both time-consuming and costly. Here, we provide a detailed protocol for efficient viral transduction of intestinal organoids. This approach is rapid and highly efficient, thus decreasing the time and expense inherent in CRISPR/Cas9 technology. We also present a protocol to generate frozen sections from intact organoid cultures for further analysis with immunohistochemical or immunofluorescent staining, which can be used to confirm gene expression or silencing. After successful transduction of viral vectors for gene expression or silencing is achieved, intestinal stem cell and crypt function can be rapidly assessed. Although most organoid studies employ in vitro assays, organoids can also be delivered to mice for in vivo functional analyses. Moreover, our approaches are advantageous for predicting therapeutic responses to drugs because currently available therapies generally function by modulating gene expression or protein function rather than altering the genome.

We describe step-by-step instructions to: 1) efficiently engineer intestinal organoids using magnetic nanoparticles for lenti- or retroviral transduction, and, 2) generate frozen sections from engineered organoids. This approach provides a powerful tool to efficiently alter gene expression in organoids for investigation of downstream effects.

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to ...

Developmental Biology

A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer

CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In higher eukaryotes, paralogous genes can mask a potential phenotype by compensating the loss of a gene, thus limiting the information that can be obtained from genetic studies relying on single gene knockouts. We have developed a novel, rapid cloning method for guide RNA (gRNA) concatemers in order to create multi-gene knockouts following a single round of transfection in mouse small intestinal organoids. Our strategy allows for the concatemerization of up to four individual gRNAs into a single vector by performing a single Golden Gate shuffling reaction with annealed gRNA oligos and a pre-designed retroviral vector. This allows either the simultaneous knockout of up to four different genes, or increased knockout efficiency following the targeting of one gene by multiple gRNAs. In this protocol, we show in detail how to efficiently clone multiple gRNAs into the retroviral CRISPR-concatemer vector and how to achieve highly efficient electroporation in intestinal organoids. As an example, we show that simultaneous knockout of two pairs of genes encoding negative regulators of the Wnt signaling pathway (Axin1/2 and Rnf43/Znrf3) renders intestinal organoids resistant to the withdrawal of key growth factors.

This protocol describes the steps for cloning multiple single guide RNAs into one guide RNA concatemer vector, which is of particular use in creating multi-gene knockouts using CRISPR/Cas9 technology. The generation of double knockouts in intestinal organoids is shown as a possible application of this method.

CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In ...

Bioengineering

LEGO: A Human GBM Organoid Model Revealing Lipid Dysregulation and Drug Targets

Laboratory-Engineered Glioblastoma Organoid Culture and Drug Screening

Glioblastoma (GBM) is described as a group of highly malignant primary brain tumors and stands as one of the most lethal malignancies. The genetic and cellular characteristics of GBM have been a focal point of ongoing research, revealing that it is a group of heterogeneous diseases with variations in RNA expression, DNA methylation, or cellular composition. Despite the wealth of molecular data available, the lack of transferable pre-clinic models has limited the application of this information to disease classification rather than treatment stratification. Transferring the patients' genetic information into clinical benefits and bridging the gap between detailed descriptions of GBM, genotype-phenotype associations, and treatment advancements remain significant challenges. In this context, we present an advanced human GBM organoid model, the Laboratory Engineered Glioblastoma Organoid (LEGO), and illustrate its use in studying the genotype-phenotype dependencies and screening potential drugs for GBM. Utilizing this model, we have identified lipid metabolism dysregulation as a critical milestone in GBM progression and discovered that the microsomal triglyceride transfer protein inhibitor Lomitapide shows promise as a potential treatment for GBM.

Here, we outline a comprehensive protocol for generating and utilizing laboratory-engineered glioblastoma organoids (LEGO) to investigate genotype-phenotype dependencies and screen potential drugs for glioblastoma treatment.

Glioblastoma (GBM) is described as a group of highly malignant primary brain tumors and stands as one of the most lethal malignancies. The genetic and ...

Cancer Research

Genetic Modification of Primate Cerebral Organoids

Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of higher-order cognitive abilities in mammals, in particular, primates. Studying gene functions in primate brains is challenging due to technical and ethical reasons, but the establishment of the brain organoid technology has enabled the study of brain development in traditional primate models (e.g., rhesus macaque and common marmoset), as well as in previously experimentally inaccessible primate species (e.g., great apes), in an ethically justifiable and less technically demanding system. Moreover, human brain organoids allow the advanced investigation of neurodevelopmental and neurological disorders.
As brain organoids recapitulate many processes of brain development, they also represent a powerful tool to identify differences in, and to functionally compare, the genetic determinants underlying the brain development of various species in an evolutionary context. A great advantage of using organoids is the possibility to introduce genetic modifications, which permits the testing of gene functions. However, the introduction of such modifications is laborious and expensive. This paper describes a fast and cost-efficient approach to genetically modify cell populations within the ventricle-like structures of primate cerebral organoids, a subtype of brain organoids. This method combines a modified protocol for the reliable generation of cerebral organoids from human-, chimpanzee-, rhesus macaque-, and common marmoset-derived induced pluripotent stem cells (iPSCs) with a microinjection and electroporation approach. This provides an effective tool for the study of neurodevelopmental and evolutionary processes that can also be applied for disease modeling.

Author Spotlight: Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification  

The electroporation of primate cerebral organoids provides a precise and efficient approach to introduce transient genetic modification(s) into different progenitor types and neurons in a model system close to primate (patho)physiological neocortex development. This allows the study of neurodevelopmental and evolutionary processes and can also be applied for disease modeling.

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of ...

CRISPR/Cas9 Gene Knockout in Sliced Human Cortical Organoids via Electroporation

Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function

Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. Studies analyzing gene function by overexpression or knockout have been instrumental in animal models to provide mechanistic insights into the regulation of neocortex development. Here, we present a detailed protocol for CRISPR/Cas9-mediated acute gene knockout by electroporation of sliced human cortical organoids. The slicing of cortical organoids aids the identification of ventricle-like structures for injection and subsequent electroporation, making this a particularly well-suited model for acute genetic manipulation during human cortical development. We describe the design of guide RNAs and the validation of targeting efficiency in vitro and in cortical organoids. Electroporation of cortical organoids is performed at mid-neurogenic stages, enabling the targeting of most major cell classes in the developing neocortex, including apical radial glia, basal progenitor cells, and neurons. Taken together, the electroporation of sliced human cortical organoids represents a powerful technique to investigate gene function, gene regulation, and cell morphology during cortical development.

Here, we present the acute genetic manipulation of sliced human cortical organoids by electroporation. These cortical organoid models are particularly amenable to injection as ventricle-like structures can be readily identified after slicing, enabling the functional investigation of human cortical development, neurodevelopmental disorders, and cortical evolution.

Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Biology

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Establishment, Maintenance, Differentiation, Genetic Manipulation, and Transplantation of Mouse and Human Lacrimal Gland Organoids

The lacrimal gland is an essential organ for ocular surface homeostasis. By producing the aqueous part of the tear film, it protects the eye from desiccation stress and external insults. Little is known about lacrimal gland (patho)physiology because of the lack of adequate in vitro models. Organoid technology has proven itself as a useful experimental platform for multiple organs. Here, we share a protocol to establish and maintain mouse and human lacrimal gland organoids starting from lacrimal gland biopsies. By modifying the culture conditions, we enhance lacrimal gland organoid functionality. Organoid functionality can be probed through a "crying" assay, which involves exposing the lacrimal gland organoids to selected neurotransmitters to trigger tear release in their lumen. We explain how to image and quantify this phenomenon. To investigate the role of genes of interest in lacrimal gland homeostasis, these can be genetically modified. We thoroughly describe how to genetically modify lacrimal gland organoids using base editors-from guide RNA design to organoid clone genotyping. Lastly, we show how to probe the regenerative potential of human lacrimal gland organoids by orthotopic implantation in the mouse. Together, this comprehensive toolset provides resources to use mouse and human lacrimal gland organoids to study lacrimal gland (patho)physiology.

This protocol describes how to establish, maintain, genetically modify, differentiate, functionally characterize, and transplant lacrimal gland organoids derived from primary mouse and human tissue.

The lacrimal gland is an essential organ for ocular surface homeostasis. By producing the aqueous part of the tear film, it protects the eye from ...

Rat Intestinal Crypts Isolation and Intestinal Organoids Generation

Generation and Manipulation of Rat Intestinal Organoids

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. Accordingly, patient-derived organoids are used for disease modeling, drug discovery, and personalized treatment screening. Mouse intestinal organoids are commonly utilized to understand aspects of both intestinal function/physiology and stem cell dynamics/fate decisions. However, in many disease contexts, rats are often preferred over mice as a model due to their greater physiological similarity to humans in terms of disease pathophysiology. The rat model has been limited by a lack of genetic tools available in vivo, and rat intestinal organoids have proven fragile and difficult to culture long-term. Here, we build upon previously published protocols to robustly generate rat intestinal organoids from the duodenum and jejunum. We provide an overview of several downstream applications utilizing rat intestinal organoids, including functional swelling assays, whole mount staining, the generation of 2D enteroid monolayers, and lentiviral transduction. The rat organoid model provides a practical solution to the need of the field for an in vitro model which retains physiological relevance to humans, can be quickly genetically manipulated, and is easily obtained without the barriers involved in procuring human intestinal organoids.

Author Spotlight: Generation and Manipulation of Rat Intestinal Organoids  

Here, we present a protocol to generate rat intestinal organoids and use them in several downstream applications. Rats are often a preferred preclinical model, and the robust intestinal organoid system fills the need for an in vitro system to accompany in vivo studies.

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. ...

CRISPR Concatemer-Mediated Multiple Gene Knockout: A Technique to Simultaneously Knockout Multiple Genes by Non-Homologous End-Joining Pathway in Mouse Intestinal Cells

Source: Merenda, A. et al., <a href="https://www.jove.com/v/55916">A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer.</a> J. Vis. Exp. (2017).
This video describes a gene knockout technique using a CRISPR-concatemer to simultaneously knock out multiple genes in cultured mouse intestinal organoid cells. This method is used to knock out a diseased gene and to elucidate the function of a gene and its paralogues.

Source: Merenda, A. et al., A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer. J. Vis. Exp. (2017).
This ...