协议的慢病毒转导与肠组织体下游分析
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
肠上皮细胞是最迅速增殖的身体组织，这导致它吸引了来自研究癌症和干细胞广泛兴趣之一。在2009年的技术被发布到基质胶产生的小肠隐窝持久的文化，涵养一个三维结构1。这些结构中，称为肠组织体，可以使用标准技术进行培养，与周围的介质补充有若干所定义的生长因子，包括BMP-信号通路抑制剂头发生素（木栓）中，Wnt信号通路的增强rspondin 1（Rspo1）和表皮生长因子（EGF）的所有发现增强肠道增殖2-4。
类器官超越了传统方面的癌细胞系，他们都是非突变，保持干细胞的层次结构，显示完整的细胞极化，表现出分化成新生的小INT发现的所有细胞系estinal上皮。因为它们可被转导携带的转基因或RNA干扰构造5，它们被用于研究特定的遗传元件，使用转基因小鼠在成本和速度的面抵销实验。就可以利用小鼠逆转录病毒或慢病毒载体6,7进行转基因表达的组织体。由于鼠逆转录病毒，能够转导分裂细胞只8的局限性，慢病毒转导是更频繁地用于细胞，很难感染，如组织体。
病毒转导和稳定表达的转基因组织体可用于多种下游分析，包括定量的RNA分析和免疫组织化学。综合来看，从小学肠上皮细胞组织体的文化已经演变成一个常规技术，很容易没有具体的实验室要求实施，并已成为CE小说标准LL文化对肠道上皮细胞的研究。
病毒转导和组织体随后的下游分析技术是繁琐的执行，并帮助组织体实验，我们产生这个视频协议，显示培养的组织体的慢病毒转导的方法。我们还展示了如何组织体正确处理可以提高产量，因此利用RNA技术或免疫增强下游的分析性能。在协议中，从它们的小肠隐窝中衍生的组织体被单独使用，但所描述的技术可应用于结肠组织体为好。
Protocol
Representative Results
Discussion
目前的视频协议描述了从初级肠上皮而这些类器官采用定量RNA技术和免疫组化下游分析组织体的慢病毒转导。
往往是在贴壁或悬浮细胞在培养板中进行慢病毒转导。自组织体的三维结构使得它们难以通过病毒颗粒渗透，使用了若干方法来增加疗效。转导后使用CHIR99021组织体的预处理增加增殖，从而可行性。重要的是要保持小细胞群体胰蛋白酶消化后，由于单电池已减少存活?…
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).
