Name: genetic engineering of primary mouse intestinal organoids using magnetic nanoparticle transduction viral vectors for frozen sectioning
Uploaded: 2019-05-10T12:30:00
Description: Watch this Scientific Journal Video about Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning at JoVE.com

This work was supported by grants from the National Institute of Health (R01DK102943, R03CA182679, R03CA191621), the Maryland Stem Cell Research Fund (2015-MSCRFE-1759, 2017-MSCRFD-3934), the American Lung Association, the Allegheny Health Network - Johns Hopkins Research Fund and the Hopkins Digestive Diseases Basic Research Core Center.

This protocol was approved by the Johns Hopkins Medical Institutions Animal Care and Use Committee (IACUC). This protocol is modified from a previously published methods10,11,12,13.
1. Preparation of Reagents
<ol>
	<li>Prepare fresh 293T medium several hours in advance and warm to 37 &#176;C in a water bath for at least 10 min before use (Table 1)....

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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=Controlled+gene+expression+in+primary+Lgr5+organoid+cultures.">Controlled gene expression in primary Lgr5 organoid cultures.</a> Nature Methods. 9 (1), 81-83 (2012).</li><li>Kabiri, Z., 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=Stroma+provides+an+intestinal+stem+cell+niche+in+the+absence+of+epithelial+Wnts.">Stroma provides an intestinal stem cell niche in the absence of epithelial Wnts.</a> Development. 141, 2206-2215 (2014).</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>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=Forskolin-induced+Swelling+in+Intestinal+Organoids:+An+In+Vitro+Assay+for+Assessing+Drug+Response+in+Cystic+Fibrosis+Patients.">Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients.</a> J Vis Exp. (120), (2017).</li><li>Mu&#241;oz, 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=The+Lgr5+intestinal+stem+cell+signature:+robust+expression+of+proposed+quiescent+'+4'+cellmarkers.">The Lgr5 intestinal stem cell signature: robust expression of proposed quiescent '+4' cellmarkers.</a> EMBO J. 31 (14), 3079-3091 (2012).</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 (2014).</li><li>Andersson-Rolf, A., Fink, J., Mustata, R. C., Koo, B. 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(90), e51765 (2014).</li><li>Sato, T., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+mouse+small+intestinal+epithelial+cell+cultures.">Primary mouse small intestinal epithelial cell cultures.</a> Methods Mol Biol. 945, 319-328 (2013).</li><li>Ye, Z., Yu, X., Cheng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+gene+transduction+of+mouse+and+human+stem+cells.">Lentiviral gene transduction of mouse and human stem cells.</a> Methods Mol Biol. 430, 243-253 (2008).</li><li>Cheung, K. J., Gabrielson, E., Werb, Z., Ewald, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+invasion+in+breast+cancer+requires+a+conserved+basal+epithelial+program.">Collective invasion in breast cancer requires a conserved basal epithelial program.</a> Cell. 155 (7), 1639-1651 (2013).</li><li>Tesfaye, 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=The+High-Mobility+Group+A1+gene+up-regulates+Cyclooxygenase-2+expression+in+uterine+tumorigenesis.">The High-Mobility Group A1 gene up-regulates Cyclooxygenase-2 expression in uterine tumorigenesis.</a> Cancer Res. 67 (9), 3998-4004 (2007).</li><li>Haim, H., 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=Synchronized+infection+of+cell+cultures+by+magnetically+controlled+virus.">Synchronized infection of cell cultures by magnetically controlled virus.</a> J. 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Primary culture of adult intestinal epithelium as organoids provides a powerful tool to study molecular mechanisms involved in stem cell function, intestinal epithelial homeostasis, and pathology1,2,3,4. Although CRISPR/Cas9 technology can be used to genetically engineer organoids9, it is limited by the need for extensive screening and selection based on sequence analysi...

The ability to culture mouse or human crypts cells as three dimensional (3D) organoids from the small intestines or colon over prolonged time periods provided a major breakthrough because these cultures display defining features of intestinal epithelium in vivo1,2,3. Organoids derived from primary crypts are capable of self-renewal and self-organization, exhibiting cellular functions similar to their tissues of origin. Indeed, organoids recapitulate not only the structural organization of crypts in vivo, but also many molecular features, thus providing useful tools to study normal biology and disease states. To illustrate, organoid studies have revealed novel molecular pathways involved in tissue regeneration1,2,3,4,5 as well as drugs that could enhance function in pathologic settings6,7.
The study of intestinal stem cells is of particular interest because the intestinal lining is among the most highly regenerative mammalian tissues, renewing itself every 3-5 days to protect the organism from bacteria, toxins, and other pathogens within the intestinal lumens. Intestinal stem cells (ISCs) are responsible for this remarkable regenerative capability and thus provide a unique paradigm for studying adult stem cell function1,2. Lineage-tracing experiments in mice demonstrated that isolated Lgr5-positive stem cells can be cultured to generate 3D organoids or &#39;mini-guts&#39; in vitro where they closely mirror their in vivo counterparts. Organoid cultures can also be derived from intestinal crypt cell isolates comprised of progenitors, ISCs, and Paneth cells, the latter of which constitute the epithelial niche cells in vivo. In fact, organoid culture from primary intestinal crypt cells has evolved into a relatively routine technique that is easy to implement in most laboratories using widely available reagents. This model is also amenable to quantitative analysis of gene expression by RNA-sequencing (RNA-Seq) and proteins by mass spectrometry, immunohistochemistry, or immunofluorescent staining2,4,8. In addition, functional genetics can be studied using gain-of-function (gene overexpression or expression of an activating mutant gene) or loss-of-function (gene silencing or expression of a loss-of-function mutant) approaches2.
Importantly, low efficiency and high toxicity of standard plasmid DNA or viral transduction protocols with polybrene remain a major hurdle in the field. Although CRISPR/Cas9 technology allows for precise genome editing, this approach requires time-consuming selection followed by sequence validation9. Here, we present a viral transduction protocol for primary intestinal organoids that optimizes delivery of viral particles by conjugation to magnetic nanoparticles and application of a magnetic field. Key modifications to prior protocols4,5,10,11,12,13 and recommendations to enhance efficiency are provided. We also describe an approach to generate frozen sections from intact organoids cultured in 3D matrigel (henceforth referred to as basement membrane matrix or matrix) for further analysis with immunohistochemistry or immunofluorescent staining.

<table>
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>11965092</td>
			<td>Base medium for 293T cells</td>
		</tr>
		<tr>
			<td>DMEM/F12+</td>
			<td>Thermo Fisher Scientific</td>
			<td>12634010</td>
			<td>Base medium for organoid culture medium and organoid digestion buffer</td>
		</tr>
		<tr>
			<td>OPTI-MEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>11058021</td>
			<td>Virus plasmids transfection medium</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Corning</td>
			<td>35-011-CV</td>
			<td>Component of virus collection medium and 293T medium</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>Component of organoid culture medium and crypt dissociation buffer</td>
		</tr>
		<tr>
			<td>PBS (without Ca2+, Mg2+)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010049</td>
			<td>A wash buffer and component of crypt dissociation buffer</td>
		</tr>
		<tr>
			<td>Mem-NEAA</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140050</td>
			<td>Component of organoid culture medium</td>
		</tr>
		<tr>
			<td>GlutamaxII</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td>Component of organoid culture medium</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630080</td>
			<td>Component of organoid culture medium</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Millipore Sigma</td>
			<td>E9644</td>
			<td>Component of organoid culture medium</td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>Peprotech</td>
			<td>250-38 B</td>
			<td>Component of organoid culture medium</td>
		</tr>
		<tr>
			<td>R-spondin</td>
			<td>R&#38;D</td>
			<td>7150-RS-025/CF</td>
			<td>Component of organoid culture medium</td>
		</tr>
		<tr>
			<td>Human recombinant insulin</td>
			<td>Millipore Sigma</td>
			<td>I9278-5ml</td>
			<td>Component of organoid culture medium</td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Millipore Sigma</td>
			<td>N3376-100G</td>
			<td>Component of Transduction medium</td>
		</tr>
		<tr>
			<td>Wnt3A</td>
			<td>R&#38;D</td>
			<td>5036-WN-010</td>
			<td>Component of Transduction medium</td>
		</tr>
		<tr>
			<td>Y27632</td>
			<td>Millipore Sigma</td>
			<td>Y0503-1MG</td>
			<td>Component of Transduction medium</td>
		</tr>
		<tr>
			<td>0.5M EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15575020</td>
			<td>Component of Crypts dissociation buffer</td>
		</tr>
		<tr>
			<td>DTT (dithiothreitol)</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0861</td>
			<td>Component of Crypts dissociation buffer</td>
		</tr>
		<tr>
			<td>Dispase I</td>
			<td>Millipore Sigma</td>
			<td>D4818-2MG</td>
			<td>Component of organoid digestion buffer</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Millipore Sigma</td>
			<td>11284932001</td>
			<td>Component of organoid digestion buffer</td>
		</tr>
		<tr>
			<td>matrigel(Growth factor reduced)</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Used as a matrix to embed organoids</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985070</td>
			<td>Medium for transfection in viral production</td>
		</tr>
		<tr>
			<td>ViralMag R/L</td>
			<td>Oz Biosiences</td>
			<td>RL40200</td>
			<td>Magnetic particles of viral transduction</td>
		</tr>
		<tr>
			<td>Magnetic plate</td>
			<td>Oz Biosiences</td>
			<td>MF10000</td>
			<td>Magnetic plate to facilitate viral transduction</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Thermo Fisher Scientific</td>
			<td>11668019</td>
			<td>Transfection agent in viral production</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Millipore Sigma</td>
			<td>A-003-E</td>
			<td>Coating for plates before seeding 293T cells</td>
		</tr>
		<tr>
			<td>4% Formaldehyde Solution</td>
			<td>Boster</td>
			<td>AR1068</td>
			<td>Solution to fix organoids</td>
		</tr>
		<tr>
			<td>O.C.T embedding compound</td>
			<td>Thermo Fisher Scientific</td>
			<td>4583S</td>
			<td>For embedding of the the organoids</td>
		</tr>
		<tr>
			<td>5 mL Falcon polystyrene tubes</td>
			<td>Corning</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Falcon Tubes</td>
			<td>Sarstedt</td>
			<td>62.547.100</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbitron rotator II Rocker Shaker</td>
			<td>Boekel Scientific</td>
			<td>260250</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus Inverted microscop CK30</td>
			<td>Olympus</td>
			<td>CK30</td>
			<td>for scanning and counting crypts</td>
		</tr>
		<tr>
			<td>Zeiss Axiovert 200 inverted fluorescence</td>
			<td>Nikon</td>
			<td>Axiovert 200</td>
			<td>for viewing fluorescence in the crypts</td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 Centrifugal Filter unit with Ultracel-100 membrane</td>
			<td>Milipore Sigma</td>
			<td>UFC910024</td>
			<td>For concentrating viruses</td>
		</tr>
		<tr>
			<td>pluriStrainer 20 &#181;m (Cell Strainer)</td>
			<td>pluriSelect</td>
			<td>SKU 43-50020</td>
			<td>For preparing organoid fragments</td>
		</tr>
		<tr>
			<td>Falcon Cell Strainer</td>
			<td>Fisher Scientific</td>
			<td>352340</td>
			<td>For preparing cyrpts of similar size after crypt isolation</td>
		</tr>
		<tr>
			<td>Greiner CELLSTAR&#160;multiwell culture plates 48 wells (TC treated with lid)</td>
			<td>Millipore Sigma</td>
			<td>M8937-100EA</td>
			<td>ForD2:D37+D16:D37g organoid fragments</td>
		</tr>
		<tr>
			<td>Animal strain: C57BL/6J</td>
			<td>Jackson Lab</td>
			<td>#000664</td>
			<td>For organoid culture</td>
		</tr>
	</tbody>
</table>

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.

Here, we describe a rapid and highly efficient transduction technique which harnesses magnetic nanoparticles exposed to a magnetic field to deliver lentivirus to cells of interest. With readily available tools, we have applied this approach not only to transduce freshly isolated crypt cells (Figure 1A), but also for organoids (Figure 2) and other cells that are refractory to more routine transduction approaches. Lentiviral partic...

Watch this Scientific Journal Video about Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning at JoVE.com

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

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 ...

Because organoids recapitulate the native structural organization, as well as many molecular features of intestinal epithelium in vivo, this technique allows us to study normal biology and disease states in the laboratory. The main advantage of this technique is the ability to genetically engineer organoids using a very highly efficient transduction approach with low cytotoxicity, thus allowing for functional studies of these genetically manipulated organoids. The implications of this technique extend toward therapy for intestinal disease because we can expose intestinal organoids to drugs and other agents or interventions.This method can be applied to study developmental and pathologic processes in other systems amenable to organoid cultures, such as breast tissues or for cells growing under standard 2D conditions. Generally, individuals new to this method will succeed with transduction in difficult-to-engineer organoids or other cells. We first had the idea for this method when we encountered difficulties transducing our organoids and stem cells.Visual demonstration of this method is critical as structural loss of the organoids can occur if samples are not properly treated during the embedding process into the basement matrix for cryosectioning. To grow organoids for viral transduction, seed organoid cell cultures with 200 microliters of transduction medium per well in a 48-well plate, and incubate in a standard tissue culture incubator. Thaw vials of virus for transduction, allowing for about 50 microliters of concentrated virus for the transduction of each well in 48-well plates.In a 1.5-milliliter tube, mix the virus with 12 microliters of a magnetic nanoparticle solution, and incubate at room temperature for 15 minutes. After 15 minutes, add the magnetic nanoparticle solution and virus mixture to the cells to be transduced. Place the 48-well plate on a magnetic plate, and incubate in a standard tissue culture incubator for at least two hours.When the viral transfection is complete, transfer the infected organoid cell clusters and transduction medium from each well into a 1.5-milliliter tube. Centrifuge at 500 times g for five minutes. Discard the supernatant with gentle suction, and cool the tubes containing the pellet on ice for five minutes.Add 120 microliters of basement membrane matrix to each tube, and resuspend the pellet by slowly pipetting up and down. Seed 30-microliter drops of the matrix-cell mixture into a new 48-well plate. Incubate the plate at 37 degrees Celsius for five to 15 minutes until the matrix solidifies.Add transduction medium to each well, and incubate in a standard tissue culture incubator for three to four days. After three to four days, inspect the cultures under a light microscope at 10x magnification to ensure organization of cell clusters into organoid structures. Gently replace the transduction medium in each well with 250 microliters of organoid culture ENR medium.Return the plate to the incubator. Begin this procedure by removing the ENR medium by gentle suction, being careful not to perturb the basement membrane matrix. Gently wash with 500 microliters of PBS.Fix the organoids by adding one microliter of 4%paraformaldehyde solution per well, and incubate at room temperature for 30 minutes. After 30 minutes, remove the paraformaldehyde solution by suction. Gently add one microliter of PBS per well, and then remove the PBS by gentle suction.In this manner, wash twice with PBS. Remove the PBS from the second wash, and add one microliter of 30%sucrose buffer to each sample. Incubate the fixed organoids in sucrose for one hour at four degrees Celsius to dehydrate the samples.Remove the sucrose buffer by suction, and add just enough embedding compound to cover the matrix layer in each well. Incubate at room temperature for five minutes. Next, place the samples in a minus 80-degree Celsius freezer for 10 minutes or until the embedding compound turns solid and white.Place the plate with frozen embedding compound at room temperature to allow for minimal melting of the compound along the edges. Use a scalpel or spatula to separate the block from the walls of the well. Work quickly to prevent melting, using forceps to remove the matrix embedding compound block, and place it in a specimen block.Fill the mold completely with embedding compound. Freeze at minus 80 degrees Celsius for 30 minutes before using the block for sectioning. This protocol was optimized by first transducing intestinal organoids with lentiviral vectors linked to GFP.The GFP was visualized at each stage in organoid development, including early on when crypt cells organize into cyst-like structures or at later time points when organoids form buds. Successfully transduced intestinal organoids were then formalin-fixed, cryosectioned, and analyzed by immunofluorescence imaging. In this image, nuclei are stained in blue, and the green indicates epithelial cell adhesion molecules, which demarcates cell borders.The red indicates lysosome staining to identify Paneth cells. Once mastered, transducing organoids can be performed in about three hours, and organoids crysection embedding can be done in about 2 1/2 hours. When attempting this procedure, it is important to remember to work on ice when handling basement matrix reagents.After completing this procedure, immunofluorescent analysis and other methods to detect proteins or organelles can be performed to assess localization or relative abundance. This technique paves the ways for researchers to explore normal biology and disease states in vitro and also for in vivo studies if the transduced organoids or cells can be implanted into mice. After watching this video, you should have a good understanding of how to use magnetically labeled viruses to genetically engineer organoids for cryosectioning.Please don't forget that working with viruses can be extremely hazardous, and precautions such as wearing appropriate protective gear should always be undertaken when performing this procedure.

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