En protokol for Lentiviral transduktion og nedstrøms analyse af Intestinal Organoids
Summary
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.
Abstract
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.
Introduction
Tarmepitelet er en af de hurtigst prolifererende kropslige væv, hvilket har forårsaget den til at tiltrække bred interesse fra forskning i kræft og stamceller. I 2009 en teknik blev offentliggjort at generere langvarige kulturer af små tarm krypter i matrigel, bevare en 3 dimensional struktur 1. Disse strukturer, betegnet intestinale organoids, kan dyrkes under anvendelse af standardteknikker, med omgivende medium suppleret med en række definerede vækstfaktorer, herunder BMP-signalvejen inhibitor noggin (Nog), Wnt-signalvejen enhancer rspondin 1 (Rspo1) og epidermal vækstfaktor (EGF) alle at forbedre intestinal proliferation 2-4.
Organoids overgå traditionelle kræft cellelinjer i de aspekter, at de er ikke-muteret, har fastholdt stamceller hierarki, vise intakt cellulær polarisering og udviser differentiering i alle cellelinier, der findes i den spirende lille intestinal epitel. Da de kan transduceres til at bære transgener eller RNA-interferens konstruerer 5, bliver de brugt til at studere specifikke genetiske elementer, opvejer forsøg med transgene mus i facetter af omkostninger og hastighed. Transgen ekspression i organoids kan udføres under anvendelse af enten murin retroviral eller lentivirusvektorer 6,7. På grund af begrænsningerne af murine retrovira, der er i stand til at transducere mitotiske celler udelukkende 8 er lentiviral transduktion oftere anvendes til celler, der er vanskelige at inficere, såsom organoids.
Viralt transduceret og stabilt udtrykker transgene organoids kan bruges til et væld af downstream analyser, herunder kvantitative RNA analyser og immunhistokemi. Tilsammen har kultur organoids fra primære tarmepitelceller udviklet sig til en rutinemæssig teknik, der er let at gennemføre uden specifikke laboratorie krav, og er blevet den nye standard i cell kultur i forskning i tarmepitelet.
Teknikker til viral transduktion og efterfølgende downstream analyse organoids er trættende at udføre, og at hjælpe organoide eksperimenter vi genererede denne video protokol, der viser metoder til lentiviral transduktion af dyrkede organoids. Vi viser desuden, hvordan korrekt behandling af organoids kan øge udbyttet og dermed forbedre ydeevnen af downstream analyse ved hjælp RNA teknikker eller immunhistokemi. I protokollen blev organoids, der er afledt fra små tarm krypter udelukkende anvendes, selv om de teknikker, der er beskrevet kan anvendes på colon organoids så godt.
Protocol
Representative Results
Discussion
Den aktuelle video protokol beskriver lentiviral transduktion af organoids fra primær tarmepitelet og nedstrøms analyse af disse organoids der anvender kvantitative RNA teknikker og immunhistokemi.
Lentiviral transduktion udføres ofte i vedhængende eller flydende celler i kultur plader. Da tredimensionelle struktur af organoids gør dem vanskelige at trænge af virale partikler, anvendes en række af metoder til at øge effekten. Forbehandling af organoids hjælp Chir99021 øger prolifer…
Disclosures
The authors have nothing to disclose.
Acknowledgements
J. Heijmans is supported by a stipendium from the Dutch cancer foundation (KFW). G.R. van den Brink is supported by funding from the European Research Council under the European Community’s Seventh Framework Program (FP7/2007-2013)/European Research Council grant agreement number 241344 and by a VIDI grant from the Netherlands Organization for Scientific Research (GvdB).
Materials
| Reagent | Company | Cat. No. |
| Polyethylene imine | polysciences | 23966-2 |
| DMEM medium | Lonza | BE12-614F |
| Fetal calf serum | Lonza | DE14-801F |
| Penicillin-streptomycin | Invitrogen | 15140-122 |
| Glutamin | Invitrogen | 25030-024 |
| matrigel | BD | BD 356231 |
| Advanced DMEM-F12 | Gibco | 12634-010 |
| N2 | Invitrogen | 17502-048 |
| B27 | Invitrogen | 17504-044 |
| N-acetyl cysteine | Sigma | A9165-1G |
| mouse Egf | Invitrogen | PMG8045 |
| Hepes 1M | Invitrogen | 15630-056 |
| glutamax 100x | Invitrogen | 35050-038 |
| Chir 99021 | axon | 1386 |
| Y27632 | Sigma | Y0503-5MG |
| polybrene | Sigma | 107689 |
| nicotinamide | Sigma | N0636 |
| Trypsin | Lonza | BE02-007E |
| puromycin | sigma | P 7255 |
| Rneasy mini kit | Qiagen | 74106 |
| b-mercaptoethanol | Merck | 8,057,400,250 |
| Ovation Pico WTA system | NuGen | 3300-12 |
| paraformaldehyde | Sigma | 252549-1L |
| glass vial conical 12mm x 75mm 5ml | VWR | LSUKM12 |
| Eosin Yellowish | VWR | 1,159,350,025 |
References
- Sato, T., et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 459 (7244), 262-265 (2009).
- Al-Nafussi, A. I., Wright, N. A. The effect of epidermal growth factor (EGF) on cell proliferation of the gastrointestinal mucosa in rodents. Virchows Archiv. B, Cell Pathology Including Molecular Pathology. 40 (1), 63-69 (1982).
- Haramis, A. P., et al. De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine. Science. 303 (5664), 1684-1686 (2004).
- Zhao, J., et al. R-spondin1, a novel intestinotrophic mitogen, ameliorates experimental colitis in mice. Gastroenterology. 132 (4), 1331-1343 (2007).
- Schwank, G., Andersson-Rolf, A., Koo, B. K., Sasaki, N., Clevers, H. Generation of BAC transgenic epithelial organoids. PLoS One. 8 (10), e76871 (2013).
- Koo, B. K., et al. Controlled gene expression in primary Lgr5 organoid cultures. Nature Methods. 9 (1), 81-83 (2012).
- Heijmans, J., et al. ER stress causes rapid loss of intestinal epithelial stemness through activation of the unfolded protein response. Cell Rep. 3 (4), 1128-1139 (2013).
- Roe, T., Reynolds, T. C., Yu, G., Brown, P. O. Integration of murine leukemia virus DNA depends on mitosis. The EMBO Journal. 12 (5), 2099-2108 (1993).
- Lau, W., et al. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature. 476 (7360), 293-297 (2011).
- Bahnson, A. B., et al. Centrifugal enhancement of retroviral mediated gene transfer. Journal Of Virological Methods. 54 (2-3), 131-143 (1995).
