The authors would like to thank Mark Bishop, Mathew Zverev, Victoria Marsh-Durban, Adam Blackwood and Sylvie Robine. This work was funded by a programme grant from Cancer Research UK.

上使用的所有材料的信息列于表1。下的英国内政部项目许可的授权进行所有的动物实验。 1.使用CatnB删除阿CRE和VIL-的Cre-ER T2系统<ol><li>越过小鼠品系产生10 - 阿CRE + CatnB + / +， 阿CRE + CatnB FLOX / FLOX，VIL- 的Cre-ER T2 CatnB + / +和VIL-的Cre-ER T2 CatnB FLOX 14周大的同伙/ FLOX。对于视觉重组的分析，通过β 半乳糖苷酶染色，同伙应该包含Rosa26R-lacZ报道 17。队列应控制为修饰基因和利用感应剂存在，需要队列的大小应该使用功率分析来估计。 </li><li>为了诱导对AH-Cre重组酶制备β;萘黄酮（BNF），或用于VIL-Cre的ER T2转基因他莫昔芬（TAM）在玉米油中，得到10毫克/毫升的工作溶液。注：代理应使用适当的个人保护通风橱中称出。 </li><li>在琥珀色瓶中热解决方案（BNF对光敏感），要么99.9℃，BNF或80℃TAM在水浴。 </li><li>转移到一个加热搅拌器设定在100℃的BNF，或80℃TAM，并搅拌10分钟。 </li><li>对于BNF重复1.3-1.4直至溶解（可以采取&gt; 1小时）。 </li><li>分装成小琥珀色瓶（〜5ml）中，然后冷冻于-20℃。瓶应后3次冷冻/解冻循环被丢弃。在使用之前，融剂和再热到适当的温度，如果他们都掉了解决方案。允许冷却至&lt;37℃在注射前。 </li><li>注入小鼠腹膜内（IP）的使用剂量为80毫克/ kg，例如25克小鼠接收0.2毫升approp的riate诱导剂。对于阿CRE提供三次注射在24小时期间，VIL-的Cre-ER T2给予一次注射，每天4天。 </li></ol> 2.解剖肠道对记者可视化和免疫组织化学（IHC） <ol><li>安乐死通过颈椎脱位不符合伦理审批前麻醉鼠标。放置鼠标在仰卧位置和润湿采用70％乙醇的毛皮，纵向沿着使用剪刀中线打开腹膜内空腔。 </li><li>固定用钳子胃和切断连接到食道。轻轻拉动胃取出小肠到附录。轻轻拉动阑尾取出大肠到肛门。 </li><li>一旦肠子已分离取出胃和阑尾。使用具有钝端枪头，注射器冲洗肠用1×PBS。注：每个肠道应该是我mmediately处理用于以下描述的下游应用之一。 </li></ol>肠癌3福尔马林固定<ol><li>切肠冲洗3到同等大小的部分和标签近端，中段和远端。切每个部分1厘米块。取一小片的手术带2厘米×2厘米。放置三至五1厘米的片上到外科胶带以金字塔中间形成。 </li><li>关闭并密封围绕纵向件的磁带，给予“日志一堆”的效果。放置组织中含有大量过量的中性缓冲的福尔马林固定液，至少10倍的固定剂对组织的体积的容积的平底容器。注意：避免将组织过量管内进行固定，将其分为多个容器。 </li><li>包埋和切片之前24小时， - 将样品在4℃下至少18个。为了防止核β-catenin的损失，不解决超过24小时。固定转印组织含有大量过量的70％EtOH中，至少10倍组织体积的平底容器之后。 </li></ol>肠癌4. M​​ethacarn固定<ol><li>之前夹层制备methacarn固定剂通过组合300毫升MeOH中，加入150ml氯仿和75ml冰醋酸（4：2：1）。切冲洗肠道分为3大段近端，中段和远端。 </li><li>将每一块肠并列在一张滤纸（15厘米×15厘米），并用弹簧弓形剪刀打开它“恩面子”。放置肠和滤纸到含有methacarn为3的玻璃皿 - 24小时，在RT。固定后拿起使用镊子的肠部分的末尾。 </li><li>缭绕钳子肠道，形成“瑞士卷”，并通过轻微打开钳子，并把一个地下25针通过它固定辊。将组织中含有一拉一平底容器RGE过量的中性缓冲的福尔马林固定剂，固定剂中的至少10倍在进行处理前的体积，以组织和存储的体积为至少1小时。 </li></ol> 5.全山LacZ的可视化（改自萨尔瓦多Marjou 等18） <ol><li>通过组合熔融ralwax用矿物油在10制备蜡板：1。倒入15个厘米培养皿待冷。制备X-gal的固定剂按表1并存储在冰上。 </li><li>除去整个肠和冲洗通过用冰冷的1×PBS，作为每小组2.修正肠通过用25ml冰冷的X-gal的固定剂冲洗。 </li><li>用剪刀剪肠道进入3 - 5等分（每盘最多5）。将每个部分到蜡板和牵制两端使部分稍微拉长与肠系膜线最上面;修剪多余的肠系膜。使用弹簧弓形剪刀剪肠道纵向和引脚输出一路走来。60; </li><li>淹没板用X-gal固定液以覆盖部分，并在4℃下至少放置1小时。用25毫升吸管除去X-gal的固定剂和用30ml的1×PBS洗一次。盖板部分用30毫升30 DTT demucifying溶液 - 60分钟在RT，理想上一个摇动平台。 </li><li>用25毫升吸管取出demucifiying解决方案和洪水板用30毫升1X的PBS。使用巴斯德吸移管清洗肠道部分与1×PBS中的板以除去粘液。 </li><li>除去的1×PBS用25毫升吸管和洪水用30ml X-gal的污点。与在摇摆平台上温和搅拌暗处孵育过夜室温。 </li><li>继温育过夜检查该部分已经开发出一种蓝色/绿色染色，如果背景颜色是白色还是新鲜染色溶液可以被添加和监控直到染色发展。注：一旦部分已经染色，然后没有进一步的染色可以尝试。 </li><li>卸下X-gal的染色用25毫升吸管和洪水板用30毫升PBS中离开3分钟，轻轻摇动。取出针，并拿起，用血管钳，肠内节结束。缭绕钳子肠道，形成“瑞士卷”，通过稍微打开钳子，并把一个地下25针通过它固定辊。 </li><li>放置组织中含有大量过量的中性缓冲的福尔马林固定液，至少10倍的固定剂对组织的体积的体积的广口平底容器。包埋和切片之前将样品在4℃下至少24小时。 </li></ol> 6.提取隐窝从肠道<ol><li>隔离前20厘米小肠，按照第2放在干净解剖面肠，使用镊子和剪刀除去任何附着的脂肪/肠系膜。使用弹簧弓形剪刀纵向打开的肠道。 </li><li>使用显微镜盖玻片，女irmly刮肠腔去除绒毛和粘液。用剪刀剪肠道到〜5毫米片并转移到50毫升管用25ml 1×HBSS补充有青霉素（100U / ml）和链霉素（100单位/毫升）。 </li><li>下室温温育10分钟。通过70微米的细胞过滤网样品取出含有抗生素的培养基。 </li><li>将肠切片到一个新的50ml管中含有10ml 1X HBSS并更换盖子。 </li><li>轻轻地颠倒两次，并使其通过70微米的细胞过滤除去1倍HBSS中。通过重复6.4＆6.5三次进一步洗肠片段，并确保最终级分是比较明显。 </li><li>转移组织到一个新的50ml管中含有10ml的EDTA（8毫摩尔）/ 1×HBSS中，并在室温离开5分钟。剧烈摇晃（20 - 30倍）或涡流，通过一个70微米的细胞过滤和传递组织块传递到一个新的50ml管中含有乙二胺四乙酸（8毫米）/ 1X HBSS。注意：流过可以被丢弃，或者如果需要绒毛上皮分析保留。 </li><li>孵育组织片在冰上30分钟，摇动样品剧烈（20 - 30倍）或涡流。通过一个70微米的细胞过滤器样品，并通过保持流动，因为这包含了墓穴。 </li><li>组织块转移到一个新的50ml管中含有10ml 1X的HBSS。剧烈摇晃（20 - 30倍）或涡流，通过一个70微米的细胞过滤，并通过保持流动。重复一次，以保证肠隐窝件的最大回收。结合通过的级分和离心机的流动在300×g离心5分钟。倒掉上清并保留隐窝沉淀。注：将粒料可立即用于培养（如果适用的话）或存储在-80℃之前，标准的DNA / RNA /蛋白质提取程序。 </li></ol> 7.标准免疫组化可视化<ol><li>切5μ石蜡包埋的组织的切片米到聚L-赖氨酸（PLL）的幻灯片。注意：标准协议用​​B连环蛋白抗体染色下面给出，对于其它抗体参数列于表2。 </li><li>去蜡2个3分钟的洗涤含有新鲜二甲苯幻灯片浴场。补充水分通过含鲜滑浴通过幻灯片3分钟：100％乙醇（2个），95％乙醇和70％的乙醇，最后进入1X PBS。 </li><li>地方滑进含柠檬酸盐缓冲液（pH 6）和热99.9℃，20分钟，以检索抗原滑动浴。允许滑动冷却，然后在含有1xTBS / T为5分钟的滑动浴洗3次5分钟。从去年洗净取出幻灯片后，立即组织周围绘制一个PAP笔，并覆盖与商业过氧化物酶块或1.5％H 2 O 2（在蒸馏水中H 2 O）部分。 </li><li>孵育20分钟，在室温（RT）然后在含有新鲜的1X TBS / T 5分钟滑动浴洗3次。洗涤后荷兰国际集团盖部分，使用移液管，在5％正常兔血清（NRS）/ 1xTBS / T为在室温30分钟以阻止主抗体的非特异性疏水结合。 <ol><li> 200用5％NRS：与B-catenin的主要抗体巴斯德吸管，并覆盖部分稀释1取下NRS块。洗幻灯片中含有新鲜1xTBS / T滑动槽3×5分钟。注 - 其他抗体需要优化稀释和特异性。以确保抗体的特异性，适当无抗体和同种型对照染色应该执行。同种型对照匹配于宿主物种和主抗体的同种型。 </li></ol></li><li>可视化与任一市售的HRP检测试剂盒或与合适的荧光标记的第二抗体（ 表2）。注：开发不同长度的每个抗体，β-catenin的10 - 15秒就足够了。要优化发展的抗体第一ESTablish的可视化使用阳性对照载玻片阳性细胞，然后所需的时间长度将此所有后续幻灯片。 </li><li>洗幻灯片中含有新鲜TBS / T滑动槽3×5分钟。通过浸渍在含有苏木滑动浴中〜45秒染液幻灯片（不是必需若采用荧光标记的第二抗体）。放幻灯片到一个干净的滑动浴和冲洗用流动的自来水为约1分钟，确保苏木没有完全洗出。 </li><li>通过穿过含有增加醇浓度幻灯片浴脱水的幻灯片; 1×30秒70％乙醇，1×30秒95％乙醇，在100％EtOH中2×30秒的洗涤，在二甲苯2×2分钟。 </li><li>在使用商用安装介质盖玻片安装幻灯片。注意：如果使用荧光标记的抗体与含有DAPI标记细胞核装入介质。 </li></ol>具体Intestina 8.组织学鉴定升上皮细胞<ol><li>肠<ol><li>准备使用部分7 用表2中描述的绒毛蛋白抗体和条件部分。 </li></ol></li><li>肠内分泌细胞（Grimelius染色18,19）。 <ol><li>通过以下步骤制备7.1-7.2部分。洗涤滑动在含有超纯水进行3分钟的滑动浴。转移到幻灯片的滑动浴含有预热银溶液（见表1）和孵育在60℃下3小时。 </li><li>在45℃下除去从银溶液，并装在一滑动浴含有新制备的预热减速器溶液（表2）的幻灯片3分钟。取出幻灯片并放入含有新鲜超纯水进行3分钟的滑动浴。按照步骤7.6-7.8从第7节。 </li></ol></li><li>杯状细胞（阿尔辛蓝染色）。 <ol><li>通过下面的步骤7.1-7.2准备部分..转移幻灯片包含阿尔辛B1中幻灯片浴UE pH为2.5 5分钟，室温。 5分钟 - 下运行的水龙头吸管和地方滑动浴3卸下阿尔新蓝染色。按照步骤7.6-7.8。注：阿辛兰溶液可以保留用于进一步使用。 </li></ol></li><li> ISC的。 <ol><li>要识别ISC的后续部分9。 </li></ol></li><li>潘氏细胞。 <ol><li>以下使用表2中描述的溶菌酶抗体和条件部分7准备的部分。 </li></ol></li></ol> 9. RNA原位检测与小鼠肠道19-21 <ol><li>将福尔马林固定的肠（第3节）5微米的部分上PLL幻灯片。准备一个线性地高辛标记的RNA探针检测OLFM4表达21。脱蜡和水化部分按照第7。注：此协议应在无RNA酶的环境中进行，以防止RNA探针的降解。 </li><li>绘制吨左右问题是PAP笔，以尽量减少试剂。在幻灯片浴用6％ 的 H 2 O 2（在蒸馏水H 2 O）30分钟温育切片。在幻灯片浴用新鲜的1×PBS 3分钟洗两次。丢弃的1×PBS，并使用用4％多聚甲醛的吸移管，盖部分20分钟在冰上。 </li><li>在幻灯片浴用新鲜的1×PBS 3分钟洗两次。使用移液管盖板部分与5分钟洗涤以幻灯片浴用新鲜的1×PBS进行3分钟的蛋白酶K溶液。使用移液管定位后的部分由覆盖在4％多聚甲醛5分钟，室温。 </li><li>在幻灯片浴用DEPC处理过的洗H 2 O 2分钟。用在乙酸酐的溶液的吸移管盖部分10分钟，同时搅拌。在幻灯片浴用新鲜的1×PBS / 3分钟，随后1×盐水/ 3分钟清洗。通过含有增加醇浓度浴通过幻灯片; 1×30秒70％乙醇，1×30秒95％乙醇，在新鲜的100％2×30秒的EtOH洗涤，2倍在新鲜的二甲苯2分钟，并允许风干。 </li><li>在杂交缓冲液100并通过加热变性探针至80℃3分钟：稀释OLFM4探头1。应用100微升探针​​与每一个部分，并用封口膜覆盖，以防止滑动的脱水。在黑暗，潮湿的室内过夜孵育在65℃。 </li><li>在幻灯片浴用5×SSC，在65℃15分钟洗净。在幻灯片浴用新鲜的50％甲酰胺30分钟在65℃下洗两次各节/ 5×SSC / 1％SDS中。两次以幻灯片浴在新鲜的PBT 10分钟，第65℃和第二在RT洗部分。使用移液管盖板部分与PBT含有25微克RNA​​酶45分钟在37℃。在PBT的滑动浴中5分钟，在室温洗部分。 </li><li>在幻灯片浴用新鲜的50％甲酰胺30分钟在65℃下洗两次各节/ 5×SSC。要阻止，使用在PBT和存储10％的绵羊血清吸管在一个黑暗的，MOI盖节圣室在RT 2 - 3小时。 </li><li> 500，用10％绵羊血清中的PBT含有5mg / ml小鼠肠粉末：通过在1稀释的抗地高辛碱性磷酸酶缀合的抗体制备抗体。孵育3小时，在4℃在黑暗中在摇动平台上。降速以除去过量的肠粉末和在PBT中添加的1％绵羊血清的3倍卷上清液。 </li><li>从幻灯片用吸管取出块，并添加100微升抗体溶液，以每节，盖上封口膜和孵育在黑暗，潮湿的房间在4℃过夜。在幻灯片浴新鲜PBT 5分钟，洗净切片3倍。到框部分中的滑动浴洗3×新鲜NTMT缓冲器5分钟。 </li><li>为了形象，用移液管覆盖每个章节与BM紫色和孵育在黑暗中，在室温24 - 72小时，直到有足够强烈的色彩发展。一旦在PBT中以幻灯片浴洗部分，并通过浸入曙红1分钟染液。圣雷莫已经通过洗涤段中的滑动浴过量曙红下一个流水为3 - 5分钟。沉浸滑动二甲苯和允许空气干燥。在使用商业媒体盖玻片坐骑。 </li></ol>肠上皮的10组织学特征<ol><li>放置从固定组织（部分3和4）5微米的部分上的PLL幻灯片。分析≥25随机整（或≥50一半）从小鼠≥4每个队列用于每个以下参数的隐窝。注：为保持一致性，从每个肠道的相同位置分析隐窝（只使用近端）。 </li><li>从标准的H＆E染色切片细胞参数（除非另有说明）：墓穴数量，高度，细胞凋亡和有丝分裂。 <ol><li>地穴号码。 <ol><li>使用低能量的倍率（ 例如 4X或10X）手动计数与基底层接触隐窝数。计数≥10横向近端肠管教派离子从至少4只小鼠。 </li></ol></li><li>地穴高度。 <ol><li>使用高功率放大倍数（ 例如 20X或40X），从地下室到隐窝/绒毛轴（ 图3b）的底部手动计数细胞的数量。 </li></ol></li><li>细胞凋亡22，23。 <ol><li>方法1：使用高功率放大倍数（ 例如 20X或40X），手动计数凋亡细胞的数目在每个墓穴。凋亡细胞可以通过细胞皱缩，染色质凝聚，形成胞质小泡和凋亡小体（图3a）来鉴定。 </li><li>方法2：执行IHC染色（部分7），用于Caspase-3的使用表2所述的条件使用高功率放大倍率（例如20X或40X。）手动计数在每个隐窝阳性细胞的数量进行定量的细胞中。细胞凋亡的执行阶段。 </li></ol></li><li>有丝分裂。 <ol&gt; <li>使用高功率放大倍数（ 例如 20X或40X），手动计数有丝分裂细胞的数目在每个墓穴。有丝分裂细胞含有冷凝的DNA材料，并且通常对称和井形成（图3b）。 </li></ol></li><li>增殖。 <ol><li>执行IHC（部分7）染色为Ki-67的使用在表2中描述的条件。手动计数阳性细胞定量隐窝细胞增殖的比例。 </li></ol></li></ol></li></ol>

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The authors have nothing to disclose.

使用条件CRE-LOX的转基因小鼠进行解剖基因和细胞的功能是一种常用的方法。这些模型已被用于取得巨大成功在肠道内确定和描述的干细胞2,4-6并了解他们的疾病25的作用。为了充分利用这些模型需要系统的综合特性，使数据可被正确地解释。这些系统的一个完整的理解是难以实现的，由于基因很少是特定于一个孤细胞类型或位置，缺乏生物知识和用于诱导Cre的表达系统的效率低下的。这里介绍的方法演示如何克服通过实验设计和应用现有知识这些问题。虽然我们用这些方法来回答一个具体的研究问题的方法这里介绍的是通用的，可以利用任何研究调查穆里NE肠。 肠组织的制备 确保稳健的结果的关键步骤是在组织，需要及时和固定协议严格遵守要处理的收获和加工。因为几乎所有显著问题下游可以归因于与组织变干和/或不完整的固定相关联的伪影。时间是至关重要的，以防止组织架构和/或核酸和蛋白质的降解。不完整或过分热心的固定可导致组织化学分辨率的损失。不完全固定由于时间或部分过厚，以允许固定剂渗透可能导致分辨率可被观察为“潮标记”时的IHC分析肠隐窝内损失不充分。而且至关重要的是，固定不延长时间过长，核β-catenin的可扩散的核心，除非马上亲了cessed蜡嵌入式福尔马林固定。 潘氏细胞在ISC独特作用 此处呈现的数据显示有效帕内特细胞在隐窝再生在成人肠下列的ISC损失的重要性。然而仍有啊，华创备件ISC的是VIL-的Cre-ER T2的目标人口的可能性。田等人的26优雅表明LGR5 喜 ISC的是由LGR5 罗储备ISC的人口取代。现在看来，很可能这些ISC的都不能幸免于阿CRE系统由于存款准备金人口已被确定为分泌细胞前体6,7。在需要的时候恢复到ISC的国家支持这些分泌细胞前体成熟的潘氏细胞的重要性还有待回答。由于潘氏细胞构成了我SC利基8和ISC反应调节，以热量的摄入27和28炎症发挥作用但它仍然有可能，他们的护理功能将延伸到自己的前体。 新的方法和技术，以有效地模拟人类大肠癌 该ISC的发现导致基因目前已被用于产生新的小鼠模型中肠生物学和疾病，由Clarke等9审查调查基因和细胞中的作用的鉴定。唯一的限制这种技术是基因表达的Cre蛋白的鉴定。目前ISC的是使用基于所述LGR5基因表达模式条件性转基因小鼠经常研究。小鼠从该LGR5启动子表达的Cre已用于删除APC，最常突变的大肠癌的基因（CRC），展示出ISC作为原点25的细胞。有选择地删除其他CRC基因在这些细胞中提供深入了解疾病的发展和传播，例如，PTEN 29。进一步了解ISC功能被检索的具体消融使用人类白喉毒素受体​​LGR5表达细胞在小鼠（DTR）基因敲到LGR5座26。其他策略使用TET-O系统，它能使突变蛋白30的持续可逆的表达。使用这些工具来修改基因在不同细胞31和位置32,33（多个）是用来了解癌症发起，进展和转移34。或者使用睡美人转系统诱变是确定CRC的新的驱动程序。老鼠，技术和基因改变战略的持续发展，不断开发出更多的耐心相关机型。 新的M编制方法已经开发了用于肠道上皮细胞和ISC的表征。可使用流量来实现的上皮细胞类型的比率表征流式细胞基于外源凝集素和CD24 35的差异表达的。潜在理解ISC生物学最大的进步及其在疾病中的作用将利用体外化培养系统 36进行。这个系统可以正常的和恶性ISC的文化在3D，在那里他们复制和分化更生理相关的方式。人们希望，这些将使药物对患者样本直接测试体外，有针对性的治疗37铺平了道路。

哺乳动物肠的腔表面特征的重复隐窝和手指的单位状突起，称为绒毛，伸入内腔。该表面是上皮的连续片材而经历完整的自我更新，大约每3 - 6天1。这种动态组织支持的快速循环的干细胞群（ISC的;也称为隐窝基底柱状细胞），它最初是由它们的LGR5基因2,3的表达鉴定。在一个专门的小生境中存在这些细胞在Lieberkuhn的隐窝的底部。起初，这ISC的是快速循环的发现是不一致的普遍想法，干细胞是静止的性质。以前的LGR5 + ISC的识别它推测，静止标签的群保持单元在4位，相对于隐窝的基极，分别将ISC的1。最近的研究ħ因为现在证明主要有等效循环综合监控系统中的每个墓穴的命运与其邻国4,5调节池调和这些意见。倘若他们失去了这些可以通过一般都致力于分泌的血统，但能恢复到ISC的，如果ISC人口受损6静止细胞所取代。 ISC的邻居可以是ISC的或他们的子细胞。该ISC的产生幼稚子细胞而繁殖和分化成组成该上皮片哪些行肠腔1的专门的细胞类型。高脚杯，肠内分泌，肠，毛束和M细胞向上迁移到内腔表面，他们提供各种吸收和监管职能，然而，帕内特细胞保持在它们混合存在与ISC的隐窝的底部。在最近几年，已经证实，该幼稚daught的比例发往分泌谱系器细胞是静态的标签保留能够在受伤6,7恢复到ISC的LGR5 卤味细胞。 由于其在隐窝再生重要性优先放在理解ISC的和它的邻居，特别是帕内特细胞之间的相互作用。帕内特细胞在其中支持ISC的8小生了至关重要的作用。除了杀菌产品帕内特细胞产生信号分子激活支配ISC更新或分化的途径。以往的研究表明，当他们可以争取通过他们的女儿潘氏细胞8提供了重要的利基信号LGR5 + ISC的可能只存在。这些研究检查了正常LGR5 + ISC的，而不是在一个情况下它们被损坏且需要从LGR5 罗人口补给帕内特细胞的作用。 <p类=“jove_content”&gt;要了解肠道生物学和疾病模型我们研究用转基因小鼠模型9,10细胞和/或基因的功能作用。经常这些模型利用CRE-LOX技术有条件地修改基因（多个）9,10。 酶Cre（造成重组）重组酶是整合家族的一个位点特异性重组酶，分离自噬菌体P1上。 酶Cre催化定义34碱基对之间位点特异性重组&#39;液氧P&#39;网站（的交叉P1基因χ）。小鼠基因工程改造而包含侧翼，其在Cre重组酶的表达被切除的感兴趣的区域LoxP位点 。联的酶Cre基因到细胞或发育特异性启动子的表达允许改变到在空间方式9,10制成，这是在克服胚胎致死突变特别有用。 华创表达进一步链接到一个受体途径，可以人为地被激活，则允许时间的改变。 使用这种技术，我们在肠道上皮细胞失活的CatnB基因11。 β连环蛋白，所述CatnB基因产物，是经典Wnt信号途径，其管辖的ISC稳态的关键调节。使用这种策略的两个以前的研究产生相互矛盾的结果12,13。由FEVR 等人12的研究中，显示出干细胞和肠道内稳态损耗。而爱尔兰等人14的研究报告指出以下在细胞活力的降低隐窝-绒毛轴从表达CatnB野生型细胞重新填充。在这些研究中的主要区别是用于表达的Cre在肠上皮细胞的启动子。所述FEVR 等人，研究中使用的绒毛蛋白基因启动子相连的雌激素受体 ​​可通过施用吨激活amoxifen（VIL-的Cre-ER T2）15,16。相比之下爱尔兰等 ，所用的鼠细胞色素P450A1（CYP1A1）的基因的启动子元件驱动的Cre表达响应于生物外源性β萘黄酮（ 阿CRE）。这些不同系统的特性产生的两个假设来解释这些不同的意见。第一个是CatnB更有效地删除了ISC使用VIL-的Cre-ER T2系统相比， 阿CRE，从而降低了综合监控系统，以分复育的级别数。或者，它是由于采用了差分CatnB缺失所述分化细胞群。该VIL-的Cre-ER T2系统目标隐窝和绒毛，而阿CRE系统的所有上皮细胞只针对ISC的利基和隐窝的非潘氏细胞。这些系统提供的理想工具用于检查behav综合监控系统的IOR及其与帕内特细胞的相互作用。在这里，我们提出了基于我们如何利用这些系统来确定帕内特细胞在介导损伤17肠道反应了关键作用的几个具体的协议。

<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>Acetate buffer</td>
			<td>*</td>
			<td>*</td>
			<td>To make 100ml: 4.8ml 0.2M Acetic acid, 45.2ml 0.2M Sodium acetate &#38; 50ml distilled water</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Fisher Scientific</td>
			<td>C/0400/PB17</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic anhydride</td>
			<td>Sigma</td>
			<td>A6404</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic anhydride solution</td>
			<td>*</td>
			<td>*</td>
			<td>2 M Acetic anhydride in 0.1 M triethanolamine hydrochloride</td>
		</tr>
		<tr>
			<td>Alcian Blue</td>
			<td>Sigma</td>
			<td>A5268</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian Blue ph2.5</td>
			<td>*</td>
			<td>*</td>
			<td>To make 500ml: 15ml acetic acid, 5g Alcian Blue &#38; 485ml distilled water</td>
		</tr>
		<tr>
			<td>anti-digoxigenin alkaline phosphatase conjugated antibody</td>
			<td>Abcam</td>
			<td>ab119345</td>
			<td></td>
		</tr>
		<tr>
			<td>B(beta)-Naphthoflavone</td>
			<td>Sigma</td>
			<td>N3633</td>
			<td>BNF, inject without allowing the solution to cool too much as compound will drop out of solution. Solution can be re-used &#8211; store at -20&#176;C between uses, do not reheat more than twice.</td>
		</tr>
		<tr>
			<td>Bloxall</td>
			<td>Vector Labs</td>
			<td>SP-6000</td>
			<td></td>
		</tr>
		<tr>
			<td>BM purple</td>
			<td>Roche</td>
			<td>11442074001</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A4503</td>
			<td>Bovine serum albumin</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>C/4920/17</td>
			<td></td>
		</tr>
		<tr>
			<td>Citrate Buffer/Antigen Unmasking Solution</td>
			<td>Vector Labs</td>
			<td>H-3300</td>
			<td></td>
		</tr>
		<tr>
			<td>Corn oil</td>
			<td>Sigma</td>
			<td>C8627</td>
			<td></td>
		</tr>
		<tr>
			<td>Demucifiying solution</td>
			<td>*</td>
			<td>*</td>
			<td>For 500ml: 50ml glycerol, 50ml Tris 0.1M pH8.8, 100ml EtOH, 300ml saline (0.9% NaCl in water), DTT 1.7g.&#160; Demucifying solution can be made in advance and stored, but DTT sholud be added just before incubation (340mg/100ml).</td>
		</tr>
		<tr>
			<td>DEPC treated water</td>
			<td>Life Technologies</td>
			<td>750023</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma</td>
			<td>101509944</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>O3690</td>
			<td>0.5M</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>E/0650DF/17</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Whatman</td>
			<td>3000917</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin&#160;</td>
			<td>Sigma</td>
			<td>SLBL11382V</td>
			<td>Neutral buffered formalin</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Sigma</td>
			<td>F5786</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehye</td>
			<td>Sigma</td>
			<td>G6257</td>
			<td></td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Sigma</td>
			<td>216763</td>
			<td></td>
		</tr>
		<tr>
			<td>Haematoxylin</td>
			<td>Raymond A Lamb</td>
			<td>12698616</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14175-053</td>
			<td>HBSS (-MgCl2+; -CaCl2)</td>
		</tr>
		<tr>
			<td>Hybridisation buffer</td>
			<td>*</td>
			<td>*</td>
			<td>&#160;5&#215; SSC, 50% formamide, 5% SDS, 1 mg/ml heparin, 1 mg/ml calf liver tRNA</td>
		</tr>
		<tr>
			<td>Hydroquinone</td>
			<td>Sigma</td>
			<td>H9003</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmPACT DAB Peroxidase</td>
			<td>Vector Labs</td>
			<td>SK-4105</td>
			<td></td>
		</tr>
		<tr>
			<td>Immpress HRP Anti-Mouse IgG Kit</td>
			<td>Vector Labs</td>
			<td>MP-7402</td>
			<td></td>
		</tr>
		<tr>
			<td>Immpress HRP Anti-Rabbit IgG Kit</td>
			<td>Vector Labs</td>
			<td>MP-7401</td>
			<td></td>
		</tr>
		<tr>
			<td>Intestinal tissue powder</td>
			<td>*</td>
			<td>*</td>
			<td>The small intestines of 5 adult mice were combined and homogenised in the minimum volume of ice cold PBS. 4 volumes of ice cold acetone were added to the homogenised intestine, which was mixed thoroughly and incubated on ice for 30 minutes. This was centrifuged and the pellet was washed using ice cold acetone. This was further centrifuged and the resulting pellet spread onto filter paper and allowed to dry. Once thoroughly dry the material was ground to a fine powder using a pestle and mortar.&#160;</td>
		</tr>
		<tr>
			<td>K-ferricyanide</td>
			<td>Sigma</td>
			<td>P-3667</td>
			<td></td>
		</tr>
		<tr>
			<td>K-ferrocyanide</td>
			<td>Sigma</td>
			<td>P3289</td>
			<td></td>
		</tr>
		<tr>
			<td>Levamisole</td>
			<td>Sigma</td>
			<td>L0380000</td>
			<td></td>
		</tr>
		<tr>
			<td>Methacarn</td>
			<td>*</td>
			<td>*</td>
			<td>60% Methanol:30% Chloroform:10% Acetic acid</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>M/4000/17</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Vector Labs</td>
			<td>S-1012</td>
			<td>NGS</td>
		</tr>
		<tr>
			<td>Normal rabbit serum</td>
			<td>Dako</td>
			<td>X0902</td>
			<td>NRS</td>
		</tr>
		<tr>
			<td>NTMT</td>
			<td>*</td>
			<td>*</td>
			<td>100 mM NaCl, 100 mM Tris HCl, 50 mM MgCl2, 0.1% Tween20, 2 mM Levamisole</td>
		</tr>
		<tr>
			<td>PAP pen</td>
			<td>Vector</td>
			<td>H-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>PBT</td>
			<td>*</td>
			<td>*</td>
			<td>0.5M NaCl, 10mM TrisHCL pH7.5, 0.1% Tween 20</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>100x solutiuon.</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (10x)</td>
			<td>Fisher Scientific</td>
			<td>BP3994</td>
			<td>Dilluted 1:10 with distilled water to make 1x</td>
		</tr>
		<tr>
			<td>PLL slides</td>
			<td>Sigma</td>
			<td>P0425-72EA</td>
			<td>Poly-L-lysine microscope slides</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma</td>
			<td>P2308</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase k solution</td>
			<td>*</td>
			<td>*</td>
			<td>Dilute Proteinase K at 200 &#181;g/ml in 50 mM Tris, 5 mM EDTA.</td>
		</tr>
		<tr>
			<td>Ralwax</td>
			<td>BDH</td>
			<td>36154 7N</td>
			<td></td>
		</tr>
		<tr>
			<td>Reducer Solution</td>
			<td>*</td>
			<td>*</td>
			<td>To make 100ml: 1g Hydroquinone, 5g sodium sulphite &#38; 100ml distilled water</td>
		</tr>
		<tr>
			<td>RnaseA</td>
			<td>Sigma</td>
			<td>R6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>*</td>
			<td>*</td>
			<td>0.9% NaCl in distilled water</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma</td>
			<td>I3771</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheep serum</td>
			<td>Sigma</td>
			<td>S3772</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>Sigma</td>
			<td>S/1240/46</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver solution</td>
			<td>*</td>
			<td>*</td>
			<td>To make 100ml: 10ml Acetate buffer, 87ml distilled water, 3ml 1% silver nitrate</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Fisher Scientific</td>
			<td>S/2120/53</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma</td>
			<td>S6753</td>
			<td>NaCl</td>
		</tr>
		<tr>
			<td>Sodium sulfite</td>
			<td>Sigma</td>
			<td>239321</td>
			<td></td>
		</tr>
		<tr>
			<td>SSC</td>
			<td>Sigma</td>
			<td>93017</td>
			<td>20x saline sodium citrate</td>
		</tr>
		<tr>
			<td>Surgical tape</td>
			<td>Fisher Scientific</td>
			<td>12960495</td>
			<td></td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma</td>
			<td>T5648</td>
			<td>TAM, inject without allowing the solution to cool too much as compound will drop out of solution. Solution can be re-used &#8211; store at -20&#176;C between uses, do not reheat more than twice.</td>
		</tr>
		<tr>
			<td>TBS/T</td>
			<td>Cell Signalling</td>
			<td>#9997</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethanolamine hydrochloride</td>
			<td>Sigma</td>
			<td>T1502</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCL</td>
			<td>Invitrogen</td>
			<td>15567-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Sigma</td>
			<td>TP9416</td>
			<td></td>
		</tr>
		<tr>
			<td>VectaMount</td>
			<td>Vector Labs</td>
			<td>H-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>VectaShield Hardset mounting Medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1500</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectastain ABC Kit</td>
			<td>Vector Labs</td>
			<td>PK-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal</td>
			<td>Promega</td>
			<td>V3941</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal fixative</td>
			<td>*</td>
			<td>*</td>
			<td>2% formaldehyde, 0.1% glutaraldehyde in 1xPBS</td>
		</tr>
		<tr>
			<td>X-gal stain</td>
			<td>*</td>
			<td>*</td>
			<td>X-gal stain; 200ul X-gal (A) in 50ml solution B (0.214g MgCl2, 0.48g K-ferricyanide, 0.734g K-ferrocyanide in 500ml PBS).&#160; Solution B can be made up oin advance and stored at 4&#176;C</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Fisher Scientific</td>
			<td>X/0200/21</td>
			<td></td>
		</tr>
	</tbody>
</table>

protocols for analyzing the role of paneth cells in regenerating the murine intestine using conditional cre-lox mouse models

哺乳动物肠的上皮表面是动态组织该更新每3 - 7天。理解这一更新过程鉴定的快速循环肠干细胞（ISCS）特征在于它们的LGR5基因表达的人口。这些由静止干细胞群体，其标志是BMI-1的表达 ，能够取代他们的伤害事件的支持。调查这些人群之间的相互作用是理解他们的疾病和癌症的作用。对肠道表面隐窝内的ISC的存在，这些壁龛支持ISC在补充上皮。活性和静态ISC的之间的相互作用可能涉及其它分化的细胞小生内，如以前已经证明，LGR5 ISC的&#39;&#39;干性&#39;&#39;紧密相连到其相邻帕内特细胞的存在。使用条件CRE-LOX鼠标模型我们测试删除多数在帕内特细胞的存在或不存在活性ISC的的效果。这里，我们描述进行以表征肠和技术和分析表明，帕内特细胞在帮助恢复以下实质性损伤ISC的小生中发挥至关重要的作用。

Watch this Scientific Journal Video about Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models at JoVE.com

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

肠上皮干细胞（ISCS）是夹杂着潘氏细胞。这些细胞分化的ISC，支持综合监控系统，并提供抗菌保护后代。在这里，我们将演示如何使用条件转基因小鼠模型，建立了潘氏细胞在维持肠道上皮细胞至关重要的作用。

协议中再生的小鼠肠道使用条件潘氏细胞中的作用分析<em&gt;的Cre-LOX</em&gt;小鼠模型

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a ...

protocols-for-analyzing-role-paneth-cells-regenerating-murine

Research

JoVE Journal

Developmental Biology

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

17.5K 次观看.  Cardiff University. 肠上皮干细胞（ISCS）是夹杂着潘氏细胞。这些细胞分化的ISC，支持综合监控系统，并提供抗菌保护后代。在这里，我们将演示如何使用条件转基因小鼠模型，建立了潘氏细胞在维持肠道上皮细胞至关重要的作用。 

视频: Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

从小鼠羊水细胞多能干细胞使用转座子系统的生产

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

科研

发育生物学

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

体外模拟微重力下小鼠腔卵泡生长的研究

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

RANKL 小鼠骨髓破骨细胞培养法研究 mTORC1 在破骨细胞形成中的作用

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are facilitated by the transparency of the animal, the ability to do forward and reverse genetic assays, the relative ease of generating fluorescently labeled proteins, and the use of fluorescent dyes that can either be microinjected into the early embryo or incorporated into its food (E. coli strain OP50) to label cellular organelles (e.g. 9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#39;H-2,2&#39;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Here, we present the use of a fluorescent pH-sensitive dye that stains intestinal lysosomes, providing a visual readout of dynamic, physiological changes in lysosomal acidity in live worms. This protocol does not measure lysosomal pH, but rather aims to establish a reliable method of assessing physiological relevant variations in lysosomal acidity. cDCFDA is a cell-permeant compound that is converted to the fluorescent fluorophore 5-(and-6)-carboxy-2&#39;,7&#39;-dichlorofluorescein (cDCF) upon hydrolysis by intracellular esterases. Protonation inside lysosomes traps cDCF in these organelles, where it accumulates. Due to its low pKa of 4.8, this dye has been used as a pH sensor in yeast. Here we describe the use of cDCFDA as a food supplement to assess the acidity of intestinal lysosomes in C. elegans. This technique allows for the detection of alkalinizing lysosomes in live animals, and has a broad range of experimental applications including studies on aging, autophagy, and lysosomal biogenesis.

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

A step-by-step guide to probe loss of lysosomal acidity in the intestine of C. elegans using the pH-sensitive vital dye 5(6)-carboxy-2&#39;,7&#39;-dichlorofluorescein diacetate (cDCFDA)

评估溶酶体碱化在活的秀丽线虫中的肠道

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

成人斑马鱼损伤模型研究泼尼松对再生骨组织的影响

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...