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&#8217;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).

1.制备聚乙烯亚胺（PEI）作为转染试剂的<ol><li>溶解约150毫克的PEI入100毫升的H 2 O. </li><li>通过添加盐酸，直到解决方案变得清晰，搅拌至完全溶解调节溶液的pH值7.4。这可能需要10至60分钟，并添加水至1毫克/毫升的终浓度。 </li><li>当清晰，筛选在5毫升分装到无菌的0.22微米的过滤器和存储的解决方案，PEI在-80℃的冰柜。 </li></ol> 2.生产慢病毒颗粒的第1天: <ol><li>拆分HEK293T细胞，以60％-80％汇合162 平方厘米烧瓶或细胞系培养基大陪替氏培养皿（DMEM补充有10％FCS，1％青霉素/链霉素和2mM谷氨酰胺补充）。 </li></ol>第2天: <ol start="2"><li>制备含有45微克总质粒DNA加在一起的DNA转染溶液慢病毒载体的包装（7微克pVSVg的; 5微克PRSV转的13微克PMDL）的编码感兴趣感兴趣或shRNA基因的慢病毒载体和20微克。调整到使用的DMEM 1ml的体积。 </li><li>制备PEI的转染溶液加入90微升1mg / ml的PEI与930微升DMEM中，并孵育5分钟，在室温下进行。 </li><li>加入DNA转染的解决方案PEI。涡流或反转的次数，并孵育5分钟，在室温下，获得的DNA转染溶液。 </li><li>滴将2ml DNA转染溶液到HEK293T细胞孵育4小时，在湿润的细胞培养箱中于37℃。 </li><li> 4小时后，刷新培养基去除PEI。这是没有必要添加新介质之前洗涤细胞。 注:PEI是细胞毒性和孵育时间长于4小时可能造成伤害HEK293T细胞。 </li></ol>第4天: <ol start="7"><li>更换苏佩rnatant新的培养基。保持上清（含病毒）;这将在步骤2.10中使用。 </li><li>把上清液在15毫升烧瓶中。要去除死细胞，离心5分钟500×克。 </li><li>采用大60毫升注射器通过0.45微米的过滤器推上清。隔夜存放于4℃。 </li></ol>第5天: <ol start="10"><li>收集第二批上清液;离心和过滤器与步骤2.8-2.9。 </li><li>在超速离心90分钟，从池步2.9和超速离心管2.10和离心机上清50,000 XG。 </li><li>取出含有超速离心管非常仔细胶囊并放入层流罩，记住在离心机内的管的方向。 </li><li>打开胶囊保持超速离心管和倾析培养基小心以这样的方式，该粒料是在管的上侧。由于病毒丸粒可能难以显现，记住什么管侧的粒料将已经形成。取微量和除去介质的最后一个比特，同时小心不要搅动不透明棕色沉淀是在超速离心管的底部的侧可见。 </li><li>重悬该沉淀在500μl类器官培养基补充了10mM烟酰胺，10μMCHIR99021，10μMY27632和8微克/毫升聚凝胺。再悬浮在该介质是重要的，因为高滴度病毒用于直接转导组织体。 </li><li>可选:冻结病毒在这个媒介分装在两批250微升-80℃。 </li></ol>组织体3慢病毒转导第0天: <ol><li>拆分全0.95厘米2以及类器官两天转成新井之前的，旨在获得约50个小类器官。根据此前公布的方案1（ 图3A，B）分裂组织体。 </li><li>补充类器官用10μM的CHIR99021和10mM烟酰胺培养基，得到囊性增生过度隐窝（ 图3C）。 注:囊性隐窝将开发最好当新鲜分裂组织体生长在CHIR99021的存在。 </li></ol>第2天: <ol start="3"><li>收获组织体通过上下抽吸基质胶和介质，从而打乱了P1000微量的混合物。放置在一个15毫升管的混合物。 </li><li>使用巴斯德吸移管，其中所述远端开口已经下降了熔化进一步扰乱。离心机组织体以沉淀5分钟，在100×g下。 注:依赖于离心机的大小，​​使用不同的离心机的速度。有必要在其中找到破坏组织体从混合物中分离出的确切的速度。 </li><li>除去上清液，并加入500μl预热1X胰蛋白酶（类似于0.25％胰蛋白酶）的。悬浮类器官的胰蛋白酶和孵化3分钟在37℃水浴。 </li><li>通过加入3.5毫升细胞系培养基的灭活胰蛋白酶（DMEM补充有10％FCS，1％青霉素/链霉素和谷氨酰胺）。离心5分钟，在500×g。 </li><li>除去上清液留下沉淀在大约20微升培养基。传送在此最后少量介质的组织体成井在48孔板。 </li><li>加入高滴度慢病毒的转导中的步骤2.14，重悬描述和继续加强3.10。当遇到低转导效率，加强3.9可以添加。 注:为了有效地传导，一般加250微升的高滴度慢病毒1等分，以组织体的一个孔。这代表在本协议的第2步描述从单一生产的收获慢50％。 </li><li>任选:以提高转导效率，通过将48孔平板含有组织体在预温热离心机上32执行spinoculation6; C和旋转，在600×g离心1小时。 </li><li>把组织体病毒混合物在培养箱中孵育1小时，在37℃的培养箱中，以允许转导。 </li><li>任选:进一步提高转导效率，在一个培养箱中孵育组织体另外3小时，在37℃。 </li><li>加入1毫升组织体培养基和悬浮的组织体病毒混合物，在850×g离心转移到微量离心管中并离心以微量为5分钟以沉淀组织体。 </li><li>去除上清，悬浮颗粒在20微升冰冷的基质胶的。因为当它成为温暖的材料固化，使用由上下吹打冰冷PBS为多次冷却的吸头。 </li><li>把液滴中的阱中一个48井板中的中间和孵化在培养箱中在37℃下进行15分钟固化。 </li><li> 15分钟后，小心地加入250微升补充有类器官培养基的10mM烟酰胺，10μMCHIR99021和10μMY27632。小打乱类器官片段将组建成小囊状类器官在24小时内。 </li></ol>第5天: <ol start="16"><li>刷新介质和选择的抗生素补充（用于嘌呤霉素，使用4微克/毫升）。 </li></ol>第7天: <ol start="17"><li>更换培养基为标准类器官培养液，辅以抗生素的选择。 注意:发芽的将是完整CHIR99021停药后2-3周。选择后，组织体可生长没有选择抗生素。 </li></ol> 4.组织体RNA准备定量RT-PCR和基因芯片<ol><li>用于RNA制备，使用根据制造商的协议的商业试剂盒。从类器官取出中期和直含基质胶类器官的圆顶增加350微升缓冲RLT的，辅以β巯基乙醇。悬浮马泰人在RLT使用P1000微量移液。 </li><li>根据制造商的协议来执行进一步的RNA制备。 </li><li>任选:使用的Ovation微微WTA系统增加的RNA产量通过扩增，根据制造商的协议规定的50微克的总RNA的初始输入。 </li><li>任选:对于前RNA的微阵列的质量控制，使用真核生物总RNA纳米芯片，旨在为8.5以上（ 图5），根据制造商的协议的RNA完整性数（RIN）上的2100生物分析仪上运行样本。 </li></ol> 5.加工类器官的石蜡包埋及免疫组化<ol><li>前类器官嵌入的起始预温热的铝块具有孔配件12毫米直径的玻璃管在培养箱中在70℃下保持液态石蜡。 </li><li>采取单井与完全成熟的组织体，并删除介质，使嵌入式类器官完好。 </li><li>加入1毫升在PBS中的4％多聚甲醛直井和在4℃下的任何地方从1小时至固定过夜。 </li><li>用1ml冰冷的PBS代替多聚甲醛。 任选:在此步骤之后保持固定组织体在PBS中长达1周，在4℃。 </li><li>重悬固定组织体在1ml的PBS中，并放置到玻璃小瓶中。 </li><li>让组织体沉入底部，持续1分钟，滗PBS中并替换，用70％的乙醇，其中一对夫妇的曙红溶液液滴的溶解，使组织体的可视化在整个嵌入处理。 </li><li>留在70％乙醇组织体在室温下。 30分钟后，通过小心地倾析，能够用肉眼来可视化，因为轻微带粉红色的曙红颜色的组织体除去70％的乙醇。更换96％的乙醇嵌入解决方案。 </li><li>重复步骤5.7，每次更换与下一个嵌入解决方案。 70％乙醇，90％乙醇，96％乙醇，100％ETHA:通过以下的溶液随后通过组织体NOL，100％的乙醇，二甲苯，二甲苯等。 </li><li>倒出最后二甲苯洗净，倒入石蜡入管。把该管立即在预加热的铝块，在70℃进行30分钟，并用新的清洁石蜡代替石蜡。 </li><li>使用预温热巴斯德吸管的大开口倾石蜡起飞和吸管组织体入石蜡块模具。保持巴斯德吸管用温暖的本生灯。放置在蜡块模具的所有类器官的一小层液体石蜡。 </li><li>尽可能，尝试操纵使用加热夹层针朝蜡块模具中心的所有组织体。保持解剖针用温暖的本生灯。 </li><li>当组织体在模具中的定位是令人满意的，硬模稍微固化的石蜡层。 </li><li>浇在上面更完成的石蜡块，并添加一个标准组织包埋盒。 </li></ol>

<ol>
	<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=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li><li>Al-Nafussi, A. I., Wright, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+epidermal+growth+factor+(EGF)+on+cell+proliferation+of+the+gastrointestinal+mucosa+in+rodents.">The effect of epidermal growth factor (EGF) on cell proliferation of the gastrointestinal mucosa in rodents.</a> Virchows Archiv. B, Cell Pathology Including Molecular Pathology. 40 (1), 63-69 (1982).</li><li>Haramis, A. P., 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=De+novo+crypt+formation+and+juvenile+polyposis+on+BMP+inhibition+in+mouse+intestine.">De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine.</a> Science. 303 (5664), 1684-1686 (2004).</li><li>Zhao, 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=R-spondin1,+a+novel+intestinotrophic+mitogen,+ameliorates+experimental+colitis+in+mice.">R-spondin1, a novel intestinotrophic mitogen, ameliorates experimental colitis in mice.</a> Gastroenterology. 132 (4), 1331-1343 (2007).</li><li>Schwank, G., Andersson-Rolf, A., Koo, B. K., Sasaki, N., 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=Generation+of+BAC+transgenic+epithelial+organoids.">Generation of BAC transgenic epithelial organoids.</a> PLoS One. 8 (10), e76871 (2013).</li><li>Koo, B. 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>Heijmans, 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=ER+stress+causes+rapid+loss+of+intestinal+epithelial+stemness+through+activation+of+the+unfolded+protein+response.">ER stress causes rapid loss of intestinal epithelial stemness through activation of the unfolded protein response.</a> Cell Rep. 3 (4), 1128-1139 (2013).</li><li>Roe, T., Reynolds, T. C., Yu, G., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+murine+leukemia+virus+DNA+depends+on+mitosis.">Integration of murine leukemia virus DNA depends on mitosis.</a> The EMBO Journal. 12 (5), 2099-2108 (1993).</li><li>Lau, W., 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=Lgr5+homologues+associate+with+Wnt+receptors+and+mediate+R-spondin+signalling.">Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling.</a> Nature. 476 (7360), 293-297 (2011).</li><li>Bahnson, A. B., 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=Centrifugal+enhancement+of+retroviral+mediated+gene+transfer.">Centrifugal enhancement of retroviral mediated gene transfer.</a> Journal Of Virological Methods. 54 (2-3), 131-143 (1995).</li></ol>

The authors have nothing to disclose.

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?...

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。

<table><tbody><tr><td>Polyethylene imine</td><td>Polysciences</td><td>23966-2</td><td></td></tr><tr><td>DMEM medium</td><td>Lonza</td><td>BE12-614F</td><td></td></tr><tr><td>Fetal calf serum</td><td>Lonza</td><td>DE14-801F</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>Glutamin</td><td>Invitrogen</td><td>25030-024</td><td></td></tr><tr><td>matrigel</td><td>BD</td><td>BD 356231</td><td></td></tr><tr><td>Advanced DMEM-F12</td><td>Gibco</td><td>12634-010</td><td></td></tr><tr><td>N2</td><td>Invitrogen</td><td>17502-048</td><td></td></tr><tr><td>B27</td><td>Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-acetyl cysteine</td><td>Sigma</td><td>A9165-1G</td><td></td></tr><tr><td>mouse Egf</td><td>Invitrogen</td><td>PMG8045</td><td></td></tr><tr><td>Hepes 1 M</td><td>Invitrogen</td><td>15630-056</td><td></td></tr><tr><td>glutamax 100x</td><td>Invitrogen</td><td>35050-038</td><td></td></tr><tr><td>Chir 99021</td><td>Axon</td><td>1386</td><td></td></tr><tr><td>Y27632&amp;nbsp;</td><td>Sigma</td><td>Y0503-5MG</td><td></td></tr><tr><td>polybrene</td><td>Sigma</td><td>107689</td><td></td></tr><tr><td>nicotinamide</td><td>Sigma</td><td>N0636</td><td></td></tr><tr><td>Trypsin</td><td>Lonza</td><td>BE02-007E</td><td></td></tr><tr><td>puromycin</td><td>Sigma</td><td>P 7255</td><td></td></tr><tr><td>Rneasy mini kit</td><td>Qiagen</td><td>74106</td><td></td></tr><tr><td>&amp;#946;-mercaptoethanol</td><td>Merck</td><td>8,057,400,250</td><td></td></tr><tr><td>Ovation Pico WTA system</td><td>NuGen</td><td>3300-12</td><td></td></tr><tr><td>paraformaldehyde</td><td>Sigma</td><td>252549-1L</td><td></td></tr><tr><td>glass vial conical 12&amp;nbsp;mm x 75&amp;nbsp;mm 5&amp;nbsp;ml</td><td>VWR</td><td>LSUKM12</td><td></td></tr><tr><td>Eosin Yellowish</td><td>VWR</td><td>1,159,350,025</td><td></td></tr></tbody></table>

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.

类器官慢病毒转导 利用慢病毒颗粒类器官传导技术事先和传导过程依赖于正确处理组织体。组织体（ 图3A）进行培养，它们被破碎成单个隐窝（ 图3B）。如以前报道的，这些单隐窝，当GSK3抑制剂CHIR99021的存在下培养成为囊性隐窝9（ 图3C）。随后组织体进行胰蛋白酶处理，以允许病毒颗粒的单细胞的渗透。当转导细胞的慢...

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A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

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.

协议的慢病毒转导与肠组织体下游分析

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

a-protocol-for-lentiviral-transduction-downstream-analysis-intestinal

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

30.5K 次观看.  Academical Medical Center. 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. 

视频: A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this clinical challenge, tumor xenograft mouse models using long-established human carcinoma cell lines and many transgenic mouse models with tumors have been developed as preclinical models. They partially mimic the features of human carcinomas, but often fail to recapitulate the key aspects of human malignancies including invasion and metastasis. Thus, alternative models that better represent the malignant progression in human CRC have long been awaited.
We herein show generation of patient-derived tumor xenografts (PDXs) by subcutaneous implantation of small CRC fragments surgically dissected from a patient. The colon PDXs develop and histopathologically resemble the CRC in the patient. However, few spontaneous micrometastases are detectable in conventional cross-sections of affected distant organs in the PDX model. To facilitate the detection of metastatic dissemination into distant organs, we extracted the tumor organoid cells from the colon PDXs in culture and infected them with GFP lentivirus prior to injection into highly immunodeficient NOD/Shi-scid IL2R&#947;null (NOG) mice. Orthotopically injected PDX-derived CRC organoid cells consistently form primary tumors positive for GFP in recipient mice. Moreover, spontaneously developing micrometastatic colonies expressing GFP are notably detected in the lungs of these mice by fluorescence microscopy. Moreover, intrasplenic injection of CRC organoids frequently produces hepatic colonization. Taken together, these findings indicate GFP-labelled PDX-derived CRC organoid cells to be visually detectable during a multistep process termed the invasion-metastasis cascade. The described protocols include the establishment of PDXs of human CRC and 3D culture of the corresponding CRC organoid cells transduced by GFP lentiviral particles.

To allow highly sensitive detection of the disseminating human colorectal cancer (CRC) cells colonizing tissues, we herein show a protocol for efficient transduction of green fluorescent protein (GFP) lentiviral particles into PDX-derived CRC organoid cells prior to their injection into recipient mice, with stereo-fluorescence microscopic observation.

淋巴结慢病毒北疆-转基因 Organoids 培养的大鼠结肠肿瘤的高灵敏度检测

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this ...

科研

癌症研究

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.

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.

胃肠道有机体遗传工程的通用高效电穿孔协议

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

遗传学

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.

使用磁纳米粒子转导病毒载体进行冷冻切片的原小鼠肠道器官的遗传工程

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

发育生物学

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

该<em&gt;离体</em&gt;文化与模式识别小鼠肠道组织体受体刺激

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

免疫与感染

Adapting Gastrointestinal Organoids for Pathogen Infection and Single Cell Sequencing under Biosafety Level 3 (BSL-3) Conditions

Human intestinal organoids constitute the best cellular model to study pathogen infections of the gastrointestinal tract. These organoids can be derived from all sections of the GI tract (gastric, jejunum, duodenum, ileum, colon, rectum) and, upon differentiation, contain most of the cell types that are naturally found in each individual section. For example, intestinal organoids contain nutrient-absorbing enterocytes, secretory cells (Goblet, Paneth, and enteroendocrine), stem cells, as well as all lineage-specific differentiation intermediates (e.g.,&#160;early or immature cell types). The greatest advantage in using gastrointestinal tract-derived organoids to study infectious diseases is the possibility of precisely identifying which cell type is targeted by the enteric pathogen and to address whether the different sections of the gastrointestinal tract and their specific cell types similarly respond to pathogen challenges. Over the past years, gastrointestinal models, as well as organoids from other tissues, have been employed to study viral tropism and the mechanisms of pathogenesis. However, utilizing all the advantages of using organoids when employing highly pathogenic viruses represents a technical challenge and requires strict biosafety considerations. Additionally, as organoids are often grown in three dimensions, the basolateral side of the cells is facing the outside of the organoid while their apical side is facing the inside (lumen) of the organoids. This organization poses a challenge for enteric pathogens as many enteric infections initiate from the apical/luminal side of the cells following ingestion. The following manuscript will provide a comprehensive protocol to prepare human intestinal organoids for infection with enteric pathogens by considering the infection side (apical vs. basolateral) to perform single-cell RNA sequencing to characterize cell-type-specific host/pathogen interactions. This method details the preparation of the organoids as well as the considerations needed to perform this work under biosafety level 3 (BSL-3) containment conditions.

Adapting Gastrointestinal Organoids for Pathogen Infection and Single Cell Sequencing under Biosafety Level 3 BSL-3 Conditions

This protocol describes how to infect human intestinal organoids from either their apical or basolateral side to characterize host/pathogen interactions at the single-cell level using single-cell RNA sequencing (scRNAseq) technology.

在生物安全3级（BSL-3）条件下使胃肠道类器官适应病原体感染和单细胞测序

Human intestinal organoids constitute the best cellular model to study pathogen infections of the gastrointestinal tract. These organoids can be derived ...

病毒感染的人肠道类器官单层的钙成像

Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory activity, actin dynamics, inflammatory responses, stem cell proliferation, and many other uncharacterized cellular functions. As such, mapping calcium signaling dynamics within the intestinal epithelium can provide insight into homeostatic cellular processes and unveil unique responses to various stimuli. Human intestinal organoids (HIOs) are a high-throughput, human-derived model to study the intestinal epithelium and thus represent a useful system to investigate calcium dynamics. This paper describes a protocol to stably transduce HIOs with genetically encoded calcium indicators (GECIs), perform live fluorescence microscopy, and analyze imaging data to meaningfully characterize calcium signals. As a representative example, 3-dimensional HIOs were transduced with lentivirus to stably express GCaMP6s, a green fluorescent protein-based cytosolic GECI. The engineered HIOs were then dispersed into a single-cell suspension and seeded as monolayers. After differentiation, the HIO monolayers were infected with rotavirus and/or treated with drugs known to stimulate a calcium response. An epifluorescence microscope fitted with a temperature-controlled, humidified live-imaging chamber allowed for long-term imaging of infected or drug-treated monolayers. Following imaging, acquired images were analyzed using the freely available analysis software, ImageJ. Overall, this work establishes an adaptable pipeline for characterizing cellular signaling in HIOs.

作者聚焦：研究肠道上皮信号传导的病毒破坏 – 研究见解和未来方向 

This protocol describes an approach for performing calcium imaging in virus-infected human intestinal organoids and offers an approach to analysis.

使用基因编码的钙指示剂对病毒感染的人肠道类器官单层进行活体钙成像

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory ...

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.

逆转录病毒感染的原发性肠化培养的视频协议

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

生物学

Establishment and Genetic Manipulation of Murine Hepatocyte Organoids

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the executor for most of the liver functions, primary hepatocytes have limited proliferating capacity. This requires the establishment of ex vivo hepatocyte expansion models for liver physiological and pathological research. Here, we isolated murine hepatocytes by two steps of collagenase perfusion and established a 3D organoid culture as the 'mini-liver' to recapitulate cell-cell interactions and physical functions. The organoids consist of heterogeneous cell populations including progenitors and mature hepatocytes. We introduce the process in detailed to isolate and culture the murine hepatocytes or fetal hepatocyte to form organoids within 2-3 weeks and show how to passage them by mechanically pipetting up and down. In addition, we will also introduce how to digest the organoids into single cells for lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering. The organoids can be used for drug screens, disease modelling, and basic liver research by modeling liver biology and pathobiology.

A long-term ex vitro 3D organoid culture system was established from mouse hepatocytes. These organoids can be passaged and genetically manipulated by lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering.

协议的慢病毒转导与肠组织体下游分析

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此视频中的章节

淋巴结慢病毒北疆-转基因 Organoids 培养的大鼠结肠肿瘤的高灵敏度检测

胃肠道有机体遗传工程的通用高效电穿孔协议

使用磁纳米粒子转导病毒载体进行冷冻切片的原小鼠肠道器官的遗传工程

该

在生物安全3级（BSL-3）条件下使胃肠道类器官适应病原体感染和单细胞测序

使用基因编码的钙指示剂对病毒感染的人肠道类器官单层进行活体钙成像

使用基因编码的钙指示剂对病毒感染的人肠道类器官单层进行活体钙成像

使用基因编码的钙指示剂对病毒感染的人肠道类器官单层进行活体钙成像

逆转录病毒感染的原发性肠化培养的视频协议

小鼠肝细胞类器官的建立与遗传操作