JHN is supported by the Swiss National Foundation (SNSF 310030_146290).

小鼠繁殖并保存在乌尔姆大学（德国乌尔姆）的动物设施无特定病原体（SPF）的条件下。所有的动物实验是根据当地的动物使用和保护委员会和国家动物福利法的指导进行。 1. pCFP-OVA质粒的构建<ol><li>扩增使用引物Ova_SpeI_fw（3&#39;- GACCAACTAGTATGGAATTTTGTTTTGATGTATT-5&#39;）和Ova_ClaI_rev（3&#39;- GACCAGATCGATTAAGGGGAAACACATCTGCC-5&#39;）37使用质粒的pCI-OVA（氨苄青霉素抗性）42作为模板的完整大小的OVA基因。克隆PCR产物（ 即 OVA编码序列）转换成使用引物和载体的SpeI位和ClaⅠ位限制位点，得到OVA特异性质粒市售噬菌体中间载体。 </li><li>用质粒垫1强组成型启动子P 超一起放大CFP编码基因</sub&gt; -cCFP（氯霉素抗性）43作为模板和引物CFP_SacII_fw（3&#39;- GATCGACCGCGGTTCTTGAAGACGAAAGGGCC-5&#39;）和CFP_SpeI_rev（3&#39;- GATCGAACTAGTACCACCACCACCACCACCTTTGTAGAGTTCATCCATGC-5&#39;）37。 </li><li>克隆在CFP编码基因导入使用引物和载体37的SpeI位和SacI限制性位点的OVA特异性质粒（氨苄青霉素抗性）。这引入了从OVA编码序列在CFP编码之间6个甘氨酸残基的间隔物。 </li></ol> 2. 大肠杆菌 pCFP-OVA建设<ol><li>在旋转摇床上生长的大肠杆菌菌株DH10B于100ml的Luria BERTANI（LB）培养基中需氧的在37℃下过夜。 </li><li>混合800微升的细菌培养用200μl甘油在-80℃以创建甘油和存储。 </li><li>从甘油加入50μl大肠杆菌菌株DH10B的5ml LB培养基中，并在37℃下需氧生长它们在旋转振荡器OVErnight。 </li><li>在旋转摇传送培养在挡板1升锥形瓶中用250毫升LB培养基在37℃。 </li><li>在600nm处生长的细菌的光学密度（OD 600） 的0.5，然后冷却在冰上培养30分钟。离心细菌在5000 xg离心在4℃下10分钟。 </li><li>重悬细菌与5000 xg离心100ml冰水冷无菌H 2 O和离心机细菌的10分钟，在4℃下重悬的细菌用100ml冰冷的10％甘油和离心机细菌在5000 xg离心10分钟在4℃下。重复此最后步骤两次。 </li><li>在最后的洗涤步骤后，重悬细菌在700微升10％的甘油。冷冻在液氮中并储存50微升等分试样在-80℃。 </li><li>在冰上解冻并转移50微升等分试样到预先冷却的电穿孔杯中（2毫米电极距离）。 </li><li>添加5微升质粒DNA（100毫微克）与50微升大肠杆菌大肠杆菌 aliquoTS。与单个脉冲在25μF，200欧姆（Ω）和2.5千伏执行电穿孔。 </li><li>悬浮的细菌在1ml预热SOC培养基（5克/升酵母提取物，2g / L胰蛋白胨，10mM的氯化钠，2.5mM的氯化钾，10mM氯化镁，10mM的硫酸镁）并孵育在37℃下需氧1小时。 </li><li>在LB琼脂平板上平板菌补充有氨苄青霉素100微克/毫升（加100微升100毫克/毫升的氨苄青霉素的库存，以100毫升的LB琼脂），并孵育过夜需氧在37℃。 </li><li>接单菌落并重新条纹它们在补充有氨苄青霉素100微克/毫升的LB琼脂平皿上。隔夜孵育有氧37℃。 </li><li>接单菌落，并在10毫升LB培养基补充有氨苄青霉素100微克/毫升为质粒分离生长过夜它们。使用市售的质粒微型制备试剂盒和洗脱质粒DNA利用根据制造商30微升的dh 2 O（蒸馏水）&#39;的规程。验证通过限制性分析和DNA测序37,42,43质粒。 </li><li>设置E的阳性克隆的培养大肠杆菌 pCFP-OVA在100ml LB培养基中补充有氨苄青霉素100微克/毫升。在37℃下需氧生长于旋转摇床上过夜。 </li><li>混合800微升用200μl纯甘油和存储的细菌培养物在-80℃。 </li><li>离心过夜培养的其余部分在5000 xg离心在室温（RT）下10分钟，并且重悬沉淀在磷酸盐缓冲盐水（PBS）。放置10微升悬浮液到载玻片，并用盖玻片覆盖。 </li><li>观察样品在标准荧光显微镜来确认CFP-OVA的表达荧光（CFP激发在450nm，在480nm处发射）。分析开始放大400X的细菌。 </li><li>离心机1毫升细菌从过夜培养物在5000×g离心10分钟，室温。悬浮颗粒在40微升PBS中并在95℃下进行10分钟煮沸。 </li><li>离心样品以30,000×g离心10分钟，用上清液进行Western印迹分析37。使用稀释1兔子饲养抗OVA多克隆抗体:400。 </li></ol> 3. OT-II的生成/小鼠红<ol><li>为了产生OT-II /红小鼠37交叉与雌性/雄性小鼠表达红色荧光蛋白的男/女OT-II小鼠（两种菌株市售）。 </li></ol> 4.脾细胞分离<ol><li>通过任何卤代醚吸入安乐死通过颈脱位对OT-II /红色小鼠麻醉后（最多5％为诱导）商购获得。 </li><li>用剪刀剪下鼠标的左上腹部。根据本腹部有脾（用暗红色椭圆形状）。用剪刀和镊子将其删除。 <ol><li>提取OT-II /红小鼠脾脏并通过它通过100微米的细胞滤网定位在用1ml注射器的柱塞，以获得单细胞悬浮液的50ml管中。洗过滤两次10mL的PBS补充有1％热灭活的胎牛血清（FBS）。 </li></ol></li><li>离心细胞悬浮液在390×g离心在RT 5分钟。重悬沉淀用5ml预温裂解缓冲液（144毫氯化铵 ，17毫摩尔Tris，pH值= 7.2）的裂解红血细胞。孵育在37℃下10分钟。 </li><li>洗样品用25毫升的PBS补充有1％FBS的。离心在390×g离心在RT 5分钟。重悬沉淀在10ml PBS中补充有1％FBS的。 </li><li>算使用Neubauer计数室中的细胞。混合10微升细胞悬浮液与10微升0.4％台盼蓝溶液。孵育1-2分钟。 <ol><li>用10微升染色的细胞填充血细胞计数器室。算上在显微镜下细胞的四个1×1毫米2的广场之一腔和determin的È每平方细胞的平均数量。活细胞出现白色，死细胞呈现蓝色。为了确定细胞的数目/ ml的稀释的因子相乘细胞数。如果2-4象限被计算，通过象限数分裂。 </li></ol></li></ol> 5. CD4 + CD62 L + T细胞富集<ol><li>丰富使用阴性或阳性选择的原则，通过磁分离试剂盒CD4 + CD62L + T细胞的脾细胞。这里，使用负选择来分离CD4 + T细胞，并使用阳性选择来进一步富集的CD4 + T细胞池中的CD62L +群体。 注意:在负选择过程，细胞，这将在不分离（此处为:所有的CD4阴性细胞）标记配上磁微珠特异性抗体。在柱洗涤步骤的CD4 + T细胞会穿过柱，以便它们可以EASILY收集。在阳性选择步骤的细胞分离标以加上磁微珠适当抗体。在柱洗涤步骤的标记的细胞会留在柱上，它们可通过从磁场去除柱，并用缓冲液漂洗它被收集。 </li><li>对于阴性选择过程，悬浮10 7个总脾细胞在冷PBS中40微升补充有0.5％牛血清白蛋白（BSA）。添加与磁分离试剂盒提供的生物素标记的抗体混合物的10微升（特异性抗体结合的非CD4 + T细胞），并在冰上孵育15分钟。 <ol><li>添加冷的PBS30μl的补充有0.5％BSA和20微升抗生物素微珠。在冰上孵育20分钟。 </li><li>添加到样品10毫升的PBS补充有0.5％BSA和离心机在390×g离心5分钟。重复此步骤两次。重悬细胞于1ml冷PBS补充有0.5％的BS一个。 </li><li>将特定于磁场中的阴性选择过程的柱并用3ml冷PBS冲洗补充有0.5％BSA的到柱上。用3ml冷PBS添加细胞悬液到柱上洗3次补充有0.5％BSA的。 </li><li>在390×g离心5分钟的流出物的离心机的细胞。流出物穿过该列包含未标记的CD4 + T细胞。重悬在1ml的PBS中补充有0.5％BSA的沉淀和计数细胞。 </li><li>算使用Neubauer计数室中的细胞。混合10微升细胞悬浮液与10微升0.4％台盼蓝溶液。孵育1-2分钟。 <ol><li>用10微升染色的细胞填充血细胞计数器室。算在显微镜下的细胞中四个1×1毫米2平方一个腔室，并确定每平方细胞的平均数量。活细胞出现白色，死细胞呈现蓝色。为了确定细胞/ mL乘法CEL的数目L请稀释因子数量。如果2-4象限被计算，通过象限数分裂。 </li></ol></li></ol></li><li>对于正的选择过程中，添加每10 7预富集的CD4 + T细胞分数为10微升CD62L微珠。在冰上孵育15分钟。 <ol><li>添加到样品10毫升的PBS补充有0.5％BSA和离心机在390×g离心5分钟。重复此步骤两次。重悬在500微升冷PBS补充有0.5％BSA的。 </li><li>放置用于负选择过程的特定列中的磁场，并用冷的PBS 500微升补充有0.5％BSA的到柱上冲洗。添加细胞悬浮液上柱洗3次用500微升冷PBS补充有0.5％BSA的。 </li><li>从磁场中删除列，并将其放置在合适的收集管。加入1毫升的PBS补充有0.5％BSA的上柱并冲洗掉的 CD4 + CD62L + T细胞■通过力推活塞入列保留在列。 注:将分离的 CD4 + CD62L + T细胞现在准备用于下游应用中，例如在体内实验。根据来自CD4 + CD62L + T细胞分离试剂盒制造商的指示，在微珠没有毒性。由于小尺寸（约50纳米），它们不活化细胞，它们将不会饱和的细胞表面表位。因此，分离的细胞可以转移到免疫缺陷小鼠中诱导结肠炎37,44,45。 </li><li>算使用Neubauer计数室中的细胞。混合10微升细胞悬浮液与10微升0.4％台盼蓝溶液。孵育1-2分钟。 <ol><li>用10微升染色的细胞填充血细胞计数器室。算在显微镜下的细胞中四个1×1毫米2平方一个腔室，并确定每平方细胞的平均数量。活细胞APPEAR白色，死细胞呈现蓝色。为了确定细胞的数目/ ml的稀释的因子相乘细胞数。如果2 - 4象限被计算，通过象限数除以。 </li></ol></li></ol></li></ol> 6.感应抗原驱动结肠炎<ol><li>注入腹膜内3×10 5个的OT-II /红色的 CD4 + CD62L + T细胞到CX 3 CR1 GFP / + X RAG - / -小鼠。抗原特异性结肠炎的诱导，辖E.大肠杆菌 pCFP-OVA在灌胃或口服滴注门牙后面的剂量在每只动物100μl的PBS中1×10 8菌落形成单元（CFU）的每隔一天。 </li><li>监测，每周两次体重移植小鼠和它们的临床状况（在小鼠的粪便，粪便稠度和活动的血存在下）。 </li></ol> 7.组织样本进行组织病理学检查<ol><li>取组织样品从第È大小鼠小肠，固定在中性缓冲的福尔马林（10％）和嵌入在石蜡如前所述36,46。 </li><li>第一个切片石蜡样品，安装在幻灯片，并进行H＆E染色47,48。 </li><li>归类大肠的组织学如先前47,48出版:正常（0分）;轻度结肠炎（1分），中度结肠炎（2分）和重度结肠炎（3分）。 </li></ol> 8.隔离肠粘膜固有层细胞<ol><li>通过颈椎脱位通过任何卤代醚吸入麻醉后（最多5％为诱导）市售安乐死移植小鼠。 </li><li>将动物打倒肚皮朝上。使用镊子向前抢皮肤尿道口。用剪刀剪开皮肤沿腹股沟到胸部腹中线。拉背部的皮肤两侧。 </li><li>透过透明的腹腔壁肌肉和切开放的体腔。确定从肛门进入盲肠49结肠。切2四肢，不受任何脂肪组织。 </li><li>纵向打开使用夹层剪刀结肠。使用镊子撼结肠组织中含有25毫升的PBS培养皿补充有1％FBS的以除去碎片和粘液。切结肠到5 - 8 mm产品件。 </li><li>放置在含有20毫升洗涤缓冲液的50ml管中的结肠段。所述洗涤缓冲液包含500毫升DPBS，10mM的HEPES和5mM的乙二胺四乙酸（EDTA）。 </li><li>涡管在最大速度30秒。 </li><li>用200rpm振荡10分钟孵育的水浴结肠段管中在37℃。 </li><li>培养后，涡管在最大速度30秒。 </li><li>丢弃上清液，然后将组织样本中含有20毫升洗涤缓冲液的一个新的50ml管中。重复步骤6-9三次</li><li>丢弃在Supernatants并将组织样品中含有25毫升的PBS的培养皿。使用镊子轻轻摇动组织样品在陪替氏培养皿几秒钟以除去残余的上皮细胞。重复此步骤三次。 </li><li>切结肠组织成2×2mm的2块和在10ml罗斯韦尔园区纪念研究所（RPMI）与来自溶组织梭菌0.5毫克/毫升胶原酶类型八和10U / ml的DNA酶I的培养基中孵育消化它们。 </li><li>涡管在最大速度30秒。 </li><li>用200rpm振荡35分钟孵育的水浴结肠片段，在37℃和涡管为在最大速度30秒，每5分钟。 </li><li>在孵育结束时涡管在最大速度30秒。 </li><li>通过使它们通过设置在一个新的50ml管中70微米的细胞滤网收集消化的片段。 </li><li>通过加入20ml的DPBS和离心机在400克5分钟洗涤样品。然后悬浮ŧ他的细胞在1毫升的DPBS。 </li><li>算使用Neubauer计数室中的细胞。混合10微升细胞悬浮液与10微升0.4％台盼蓝溶液。孵育1-2分钟。 <ol><li>用10微升染色的细胞填充血细胞计数器室。算在显微镜下的细胞中四个1×1毫米2平方一个腔室，并确定每平方细胞的平均数量。活细胞出现白色，死细胞呈现蓝色。为了确定细胞的数目/ ml的稀释的因子相乘细胞数。如果2-4象限被计算，通过象限数分裂。 </li></ol></li></ol> 9.外染色的FCM分析<ol><li>离心390×g的中电细胞5分钟。重悬细胞于1ml的FACS A的（PBS补充有1％BSA和0.1％叠氮化钠） 注:叠氮化钠是剧毒。它可引起低血压，低体温，头痛抽搐甚至死亡。稀叠氮化钠溶液（0.1％至1.0％），通常不呈现非凡危险给用户，但它们的眼睛和皮肤的刺激物。因此，使用叠氮化钠只有在化学罩，而穿着实验室外套和一个双对一次性丁腈橡胶手套。 </li><li>孵育15分钟，将细胞在室温用FACS A.稀释单克隆抗体（mAb）2.4G2针对FcγRIII/ II CD16 / CD32（0.5-1微克单抗/ 10 6个细胞） </li><li>加入200μlFACS A的在390×g的细胞和离心5分钟。重复此步骤两次。孵育将细胞与0.5在4℃纳克相关mAb的/ 10 6个细胞20分钟。稀释FACS A.抗体</li><li>加入200μlFACS A的在390×g的细胞和离心5分钟。重复此步骤两次。重悬的样品在100μl的FACS A的</li><li>从CX 3分析T细胞CR1 GFP / + X RAG - / -与OT-II /红色的CD4 T细胞在流式细胞仪37的流动重组小鼠。 </li><lI&gt;使用市售软件分析数据。 </li></ol> 10.共聚焦显微镜分析<ol><li>分析重组或不与OT-II /红色的 CD4 + CD62L + T细胞的CX 3 CR1 GFP / +小鼠的结肠和口服喂养每隔一天用大肠杆菌的1×10 8 CFU 大肠杆菌菌株DH10B pCFP-OVA。 </li><li>取下用剪刀和镊子小鼠结肠。打开它使用纵向解剖剪刀。 </li><li>切割用剪刀一个5-8毫米一块结肠样品，放到玻片，用盖玻片覆盖。 </li><li>以40 / 1.30的倍率分析用共焦显微镜的结肠样品。 </li><li>检测在CX 3 CR1 +国会议员，标记有绿色荧光蛋白（GFP），使用一个滤波器与在488nm激发和发射在509处设置的。 </li><li>检测表达使用过滤器在58与554纳米的激发和发射设置红蛋白的CD4 + T细胞6纳米。 </li><li>使用过滤器以在450nm激发和发射在480nm设置检测大肠杆菌 pCFP-OVA。 </li><li>定义感兴趣的区域，并产生如先前所述50进行的共定位分析散点图。使用2种不同的公式:按曼德斯和Pearson的系数的重叠系数。 </li></ol>

The authors have nothing to disclose.

与所有其他的模式，上述的抗原驱动结肠炎模型可呈现在执行该技术的研究者必须意识到几个问题。当注射OT-II /红+ CD4 + T CD62L +细胞在主机上，研究者必须非常温柔细心针插入腹腔。不这样做可能会导致在小鼠可能导致死亡，或这将不会引起任何疾病的细胞的皮下给药的肠的撕裂。 通常情况下，老鼠会在细胞转移后的第一周体重增加。研究者应当在?...

肠道是暴露于外部环境的人体最大的表面上。居民微生物的广大阵列殖民人体的肠道内形成的肠道菌群（或微生物）。这估计是由最多百万亿微生物细胞和构成生物学1-3知人口最稠密的细菌栖息地之一。在GIT细菌定植肠道的利基在那里生存繁衍4。作为回报，菌群赋予了未编码它的基因组中1个附加的功能特征的主机。例如微生物群刺激上皮细胞的增殖，通过自身产生承载不能生产维生素，调节代谢和防止病原体4-6。鉴于这种有利的关系，一些作者认为人是"超生物体"或"holobionts"是细菌和人类基因7,8的混合。鉴于（人）主机上的微生物的有利影响，肠道免疫系统需要耐受共生微生物，使他们能够管腔存在，而且杀了，从腔侧9-11入侵的病原体。肠道免疫系统已开发机制无害的和潜在有害的管腔微生物来区分;然而这些机制尚未很好地理解12。维护肠道完整性要求严格调节免疫稳态保持宽容和豁免权13之间的平衡。在免疫稳态不平衡有助于肠道疾病的诱导如炎性肠病（IBD）3,14。 有两种主要类型的IBD:克罗恩病（CD）和溃疡性结肠炎（UC）。患者与这些疾病通常患有直肠出血，严重腹泻和腹痛15,16。 IBD的单一原因仍未知的，但遗传因素，环境因素和错调免疫反应的组合可能是疾病发展的15关键事件。 鸡传染性法氏囊病动物模型已经使用了超过50年。在过去几十年中新的IBD模型系统已被开发来测试关于IBD 17,18的发病机制中的各种假说。慢性结肠炎的最佳表征的模型是诱导T细胞稳态19,20的破坏的T细胞转移模型。该模型涉及到从免疫活性小鼠转印幼稚T细胞分化成缺乏T和B细胞（如RAG - / -和SCID小鼠）的主机16,21。疾病在该模型的发展是通过评估腹泻的存在下，体力活动减少和体重的损失为3-10周监测。这就是所谓的消耗综合征16。相比于健康小鼠移植主机的结肠组织是thickeR，短重16。使用T细胞转移模型中，也可以理解的T细胞群如何不同可以向IBD 22的发病机制。 T细胞转移模型不分析抗原特异性方式在疾病过程中APC和T细胞之间的相互作用。它已经表明，骨髓细胞和淋巴样细胞之间的相互作用可能是负责肠道炎症23的发展。尽管IBD的许多方面都得到了澄清，仍然需要清楚地理解，导致疾病发展的初始事件。 它已经表明，在不存在的微生物转移结肠炎不能建立24。最近，一些理论认为IBD可以针对共生细菌25的免疫应答的结果。作者也提出了共生菌是必不可少的诱发炎症在远端肠26，在无菌（GF）的动物的肠道免疫系统通常受损27,28，但这些小鼠中完全胜任肠道免疫系统29的发展无特定病原体动物细菌结果的混合物的定植。因此，该微生物群似乎是在IBD的发病机理的一个关键因素，无论是作为可诱发或防止肠炎症30,31的发展的机制。当前理论认为IBD是微生物不平衡，称为生态失调，遗传倾向的患者32的结果，但是它是目前尚不清楚，如果生态失调是原因或疾病12的结果。考虑到微生物在IBD的发展中的作用， 在体外实验表明，CD4 + T细胞可以通过肠道细菌33,34脉冲式装甲运兵车被激活。 此外，已经表明，从抗原不同共生细菌物种，例如大肠杆菌大肠杆菌，类杆菌，真杆菌和变形杆菌 ，能够激活CD4 + T细胞35。这表明，细菌抗原给T细胞的呈现是重要的，对IBD的发展。为了减少通过在疾病过程中的微生物衍生的多种抗原的复杂性，在大肠杆菌菌株已创建产生该OVA抗原。转移结肠炎诱导OVA特异性T细胞注射到RAG - / -动物的OVA表达E.殖民大肠杆菌 。 这种模型是基于最近的证据表明，CX 3 CR1 +国会议员，在结肠固有层（CLP）36的主要细胞亚群，与CD4 + T细胞转移过程中相互作用肠炎37。国会议员采样为颗粒性抗原肠腔，如细 ​​菌，使用他们的树突36，38,39。以往的研究表明，国会议员也可以占用可溶性抗原，如OVA，引入到肠腔40,41。给出的丰度的CX 3 CR1 +国会议员中电的，可能的是这些细胞可以品尝管腔细菌和与CD4的T细胞相互作用。小鼠共焦成像与大肠杆菌定植OVA特异性CD4 + T细胞移植大肠杆菌 CFP-OVA，表明CX 3 CR1 +国会议员都在抗原驱动结肠炎的发展过程中与OT-II CD4 + T细胞接触。这种模式使肠道装甲运兵车和具体仅在肠腔特定的抗原表达细菌性T细胞的抗原呈递过程的研究。

<table><tbody><tr><td>LB Broth, Miller (Luria-Bertani)</td><td>Difco</td><td>244620</td><td></td></tr><tr><td>Rotary Shake</td><td>Reiss Laborbedarf e. K.</td><td>Model 3020 GFL</td><td></td></tr><tr><td>2 mm gap couvettes&amp;nbsp;</td><td>Peqlab Biotechnologie GmbH</td><td>71-2020</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516-100ML</td><td></td></tr><tr><td>Gene Pulser Xcell system&amp;nbsp;</td><td>BioRad Laboratories GmbH</td><td>1652660</td><td></td></tr><tr><td>LB Agar, Miller (Luria-Bertani)</td><td>Difco</td><td>244510</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma-Aldrich</td><td>A9393-5G</td><td></td></tr><tr><td>SOC Medium</td><td>Sigma-Aldrich</td><td>S1797-100ML</td><td></td></tr><tr><td>High Pure Plasmid Isolation Kit</td><td>Roche</td><td>11754777001</td><td></td></tr><tr><td>Agarose</td><td>Carl Roth GmbH &amp;amp; Co</td><td>3810.1</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E9884-100G</td><td></td></tr><tr><td>Tris-HCl</td><td>Sigma-Aldrich</td><td>T5941</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Sigma-Aldrich</td><td>537020&amp;nbsp;</td><td></td></tr><tr><td>Gel chamber&amp;nbsp;</td><td>PEQLAB Biotechnology GmbH</td><td>40-0708</td><td></td></tr><tr><td>Loading Dye</td><td>Thermo Fisher</td><td>R0611</td><td></td></tr><tr><td>GeneRuler 1 kb DNA Ladder&amp;nbsp;</td><td>Thermo Fisher</td><td>SM0312</td><td></td></tr><tr><td>Ethidium bromide solution</td><td>Carl Roth GmbH &amp;amp; Co. KG</td><td>2218.3</td><td></td></tr><tr><td>Photo-documentation system&amp;nbsp;</td><td>Decon Science Tech GmbH</td><td>DeVision G&amp;nbsp;</td><td></td></tr><tr><td>DNA sequencing&amp;nbsp;</td><td>MWG-Biotech GmbH</td><td></td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Biochrom</td><td>L182-50</td><td></td></tr><tr><td>Fluorescent microscope&amp;nbsp;</td><td>Zeiss</td><td>HBO 100</td><td></td></tr><tr><td>Mini-PROTEAN Tetra System</td><td>Bio-Rad Laboratories GmbH</td><td>1658005</td><td></td></tr><tr><td>PageRuler Prestained Protein Ladder&amp;nbsp;</td><td>Fermentas, St. Leon-Rot, Germany</td><td></td><td></td></tr><tr><td>IstanBlue Solution</td><td>Expedeon, Cambridgeshire, United Kingdom</td><td></td><td></td></tr><tr><td>Nitrocellulose membrane&amp;nbsp;</td><td>Macherey-Nagel GmbH &amp;amp; Co. KG</td><td>741280</td><td></td></tr><tr><td>Electro blotter&amp;nbsp;</td><td>Biometra GmbH</td><td>846-015-600</td><td></td></tr><tr><td>Bovine Serum Albumins (BSA)</td><td>Sigma-Aldrich</td><td>A6003-25G</td><td></td></tr><tr><td>Anti-Ovalbumin antibody&amp;nbsp;</td><td>Abcam</td><td>ab181688</td><td></td></tr><tr><td>Anti-rabbit IgG&amp;nbsp; HRP</td><td>Sigma-Aldrich</td><td>A0545&amp;nbsp;</td><td></td></tr><tr><td>Pierce ECL Plus Western Blotting Substrate</td><td>Pierce Biotechnology, Thermo Fischer Scientific Inc</td><td>32132</td><td></td></tr><tr><td>Forene</td><td>Abbott</td><td>2594.00.00</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>10500-064</td><td></td></tr><tr><td>Falcon Cell Strainers</td><td>Fischer Scientific&amp;nbsp;</td><td>08-771-2</td><td></td></tr><tr><td>Ammonium chloride</td><td>Sigma-Aldrich</td><td>254134-5G</td><td></td></tr><tr><td>Tris Base</td><td>Sigma-Aldrich</td><td>10708976001</td><td></td></tr><tr><td>CD4&lt;sup&gt;+&lt;/sup&gt; CD62 L&lt;sup&gt;+&lt;/sup&gt; T isolation kit&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-093-227&amp;nbsp;</td><td></td></tr><tr><td>MACS LS Columns&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>MACS MS Columns&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>MidiMACS Separator</td><td>Miltenyi Biotec</td><td>130-042-302</td><td></td></tr><tr><td>MiniMACS Separator</td><td>Miltenyi Biotec</td><td>130-042-102</td><td></td></tr><tr><td>MACS MultiStand</td><td>Miltenyi Biotec</td><td>130-042-303</td><td></td></tr><tr><td>Feeding Needle 20G</td><td>SouthPointe Surgical Supply, Inc</td><td>FN-7903</td><td></td></tr><tr><td>Formalin solution, neutral buffered, 10%</td><td>Sigma-Aldrich</td><td>HT501128</td><td></td></tr><tr><td>Paraffin</td><td>Sigma-Aldrich</td><td>1496904</td><td></td></tr><tr><td>Hematoxylin</td><td>Sigma-Aldrich</td><td>H9627</td><td></td></tr><tr><td>Eosin Y</td><td>Sigma-Aldrich</td><td>230251&amp;nbsp;</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma-Aldrich</td><td>D9779&amp;nbsp;</td><td></td></tr><tr><td>Collagenase type VIII</td><td>Sigma-Aldrich</td><td>C-2139</td><td></td></tr><tr><td>Roswell Park Memorial Institute (RPMI) medium</td><td>AppliChem</td><td>A2044, 9050</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>438456</td><td></td></tr><tr><td>Mouse BD Fc Block</td><td>BD Pharmingen</td><td>553141</td><td></td></tr><tr><td>FITC-conjugated mAb binding V&amp;szlig; 5.1, 5.2&amp;nbsp;</td><td>BD Pharmingen</td><td>553189</td><td></td></tr><tr><td>APC-conjugated mAb binding CD4 GK1.5&amp;nbsp;</td><td>eBioscience</td><td>17-0041-83</td><td></td></tr><tr><td>FACS Calibur&amp;nbsp;</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>FCS Express V3 software</td><td>DeNovo</td><td></td><td></td></tr><tr><td>Meta scanning confocal microscope&amp;nbsp;</td><td>Zeiss</td><td>LSM 710&amp;nbsp;</td><td></td></tr><tr><td>Zeiss Workstation</td><td>Zeiss</td><td>LSM 7</td><td></td></tr><tr><td>Zeiss ZEM software&amp;nbsp;</td><td>Zeiss</td><td>v4.2.0.121</td><td></td></tr><tr><td>Maxisorp immuno plates&amp;nbsp;</td><td>NUNC, Roskilde</td><td>442404</td><td></td></tr><tr><td>Streptavidin conjugated alkaline phosphatase</td><td>Jackson Immuno Research</td><td>016-050-084</td><td></td></tr><tr><td>Alkaline phosphatase substrate 4-Nitrophenyl phosphate disodium salt hexahydrate</td><td>Sigma-Aldrich</td><td>71768-5G</td><td></td></tr><tr><td>mAb R4-6A2</td><td>BD Biosciences</td><td>551216</td><td></td></tr><tr><td>mAb XMG1.2&amp;nbsp;</td><td>BD Biosciences</td><td>554410</td><td></td></tr><tr><td>TECAN microplate-ELISA reader</td><td>Tecan</td><td></td><td></td></tr><tr><td>EasyWin software</td><td>Tecan</td><td></td><td></td></tr><tr><td>DNase I recombinant</td><td></td><td>4536282001</td><td></td></tr></tbody></table>

development of an antigen-driven colitis model to study presentation of antigens by antigen presenting cells to t cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

要建立抗原驱动结肠炎模型中的E.大肠杆菌菌株 已构建了包含在其中在强组成型启动子P 超 （ 图1A）的控制被表示为在CFP的基因融合到为鸡卵清蛋白和所述融合构建体的编码序列的质粒。荧光显微镜显示重组大肠杆菌大肠杆菌 pCFP-OVA，但不是亲本大肠杆菌大肠杆菌 DH10B，表达CFP（ 图1B）。 CFP-OVA-生产E.?...

Watch this Scientific Journal Video about Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells at JoVE.com

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

抗原驱动结肠炎模型的开发由抗原呈递细胞T细胞抗原研究的介绍

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

development-an-antigen-driven-colitis-model-to-study-presentation

Research

JoVE Journal

Immunology and Infection

9.0K 次观看.  University College Cork. In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

视频: Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement.1 Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu.2 While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different.3 This distinction argues the need for specific animal models of CAC.
Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors.4 Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]).5 
The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks.6, 7 Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.8

Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

建模结肠炎相关的癌症与​​氧化偶氮甲烷（AOM）和葡聚糖硫酸钠（DSS）

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal ...

科研

医学

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

免疫与感染

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

“<em&gt;枸橼酸rodentium</em&gt;鼠标产品型号：研究感染性结肠炎的病原体和宿主的贡献

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

Visualization of Estrogen Receptors in Colons of Mice with TNBS-Induced Crohn's Disease using Immunofluorescence

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis disorder and damage of intestinal mucosa and seems to be associated with an increased risk of colon neoplastic transformation. Accumulating evidence indicates that estrogens and estrogen receptors affect not only hormone-sensitive tissues, but also other tissues not directly related to estrogens, such as the lungs or colon. Here, we describe the protocol for the successful immunofluorescence staining of estrogen receptors in colon obtained from a murine model of TNBS-induced Crohn&#39;s disease. A detailed protocol for the induction of Crohn&#39;s disease in mice and intestine preparation is provided as well as a step-by-step immunohistochemical procedure using formalin-fixed paraffin-embedded intestine sections. The described methods are not only useful for estrogen receptor detection and estrogen signaling investigation in vivo but can also be applied to for other proteins which may be involved in the development of colitis.

The protocol presents a complete validated TNBS-induced murine model of Crohn&#39;s disease and methods for visualization of estrogen receptors by immunohistochemistry using immunofluorescence of formalin-fixed colon sections embedded in paraffin.

使用免疫荧光对患有TNBS诱导克罗恩病的小鼠结肠雌激素受体的可视化

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis ...

生物化学

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three&#160;weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48&#160;hr and rats fed SO by 72&#160;hr. Weight gain resumed after 72&#160;hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5&#160;days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.

DNBS/TNBS induced colitis offers an alternative and inexpensive method to study the pathobiology of inflammatory bowel disease. The protocol discussed in this paper outlines the successful application of DNBS to induce colitis in mice and rats and allows one to thoroughly study host-mediated intestinal responses.

二硝基苯磺酸/ TNBS结肠炎模型：提供洞察炎症性肠病和膳食脂肪的影响

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

IL-22表达淋巴细胞使用记者小鼠的可视化

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

小鼠远端结肠造口术, 一种新的 C57BL/6 老鼠转移性结肠炎模型

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

Investigating Target Gene Function in a CD40 Agonistic Antibody-induced Colitis Model using CRISPR/Cas9-based Technologies

The immune system functions to defend humans against foreign invaders such as bacteria and viruses. However, disorders of the immune system may lead to autoimmunity, inflammatory disease, and cancer. The inflammatory bowel diseases (IBD)-Crohn's disease (CD) and ulcerative colitis (UC)-are chronic diseases marked by relapsing intestinal inflammation. Although IBD is most prevalent in Western countries (1 in 1,000), incident rates are increasing around the world. Through association studies, researchers have linked hundreds of genes to the pathology of IBD. However, the elaborate pathology behind IBD and the high number of potential genes pose significant challenges in finding the best therapeutic targets. Additionally, the tools needed to functionally characterize each genetic association introduce many rate-limiting factors such as the generation of genetically modified mice for each gene. To investigate the therapeutic potential of target genes, a model system has been developed using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and a cluster of differentiation 40 (CD40) agonistic antibody. The present study shows that CRISPR/Cas9-mediated editing in the immune system can be used to investigate the impact of genes in vivo. Limited to the hematopoietic compartment, this approach reliably edits the resulting reconstituted immune system. CRISPR/Cas9-edited mice are generated faster and are far less expensive than traditional genetically modified mice. Furthermore, CRISPR/Cas9 editing of mice has significant scientific advantages compared to generating and breeding genetically modified mice such as the ability to evaluate targets that are embryonic lethal. Using CD40 as a model target in the CD40 agonistic antibody-induced colitis model, this study demonstrates the feasibility of this approach.

<meta charset="utf8"></head><body>使用基于 CRISPR/Cas9 的技术研究 CD40 激动抗体诱导的结肠炎模型中的靶基因功能

Here, we describe the methodology to knock out a gene of interest in the immune system using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and the evaluation of these mice in a cluster of differentiation 40 (CD40) agonistic antibody-induced colitis model.

使用基于 CRISPR/Cas9 的技术研究 CD40 激动抗体诱导的结肠炎模型中的靶基因功能

The immune system functions to defend humans against foreign invaders such as bacteria and viruses. However, disorders of the immune system may lead to ...

抗原驱动结肠炎模型的开发由抗原呈递细胞T细胞抗原研究的介绍

摘要

探索更多视频

此视频中的章节

建模结肠炎相关的癌症与氧化偶氮甲烷（AOM）和葡聚糖硫酸钠（DSS）

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

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使用免疫荧光对患有TNBS诱导克罗恩病的小鼠结肠雌激素受体的可视化

二硝基苯磺酸/ TNBS结肠炎模型：提供洞察炎症性肠病和膳食脂肪的影响

诱导小鼠肠道炎症由效应CD4的过继转移

IL-22表达淋巴细胞使用记者小鼠的可视化

小鼠远端结肠造口术, 一种新的 C57BL/6 老鼠转移性结肠炎模型

使用基于 CRISPR/Cas9 的技术研究 CD40 激动抗体诱导的结肠炎模型中的靶基因功能

使用基于 CRISPR/Cas9 的技术研究 CD40 激动抗体诱导的结肠炎模型中的靶基因功能

抗原驱动结肠炎模型的开发由抗原呈递细胞T细胞抗原研究的介绍

摘要

探索更多视频

此视频中的章节

建模结肠炎相关的癌症与​​氧化偶氮甲烷（AOM）和葡聚糖硫酸钠（DSS）

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

“

使用免疫荧光对患有TNBS诱导克罗恩病的小鼠结肠雌激素受体的可视化

二硝基苯磺酸/ TNBS结肠炎模型：提供洞察炎症性肠病和膳食脂肪的影响

诱导小鼠肠道炎症由效应CD4的过继转移

IL-22表达淋巴细胞使用记者小鼠的可视化

小鼠远端结肠造口术, 一种新的 C57BL/6 老鼠转移性结肠炎模型

使用基于 CRISPR/Cas9 的技术研究 CD40 激动抗体诱导的结肠炎模型中的靶基因功能

使用基于 CRISPR/Cas9 的技术研究 CD40 激动抗体诱导的结肠炎模型中的靶基因功能

建模结肠炎相关的癌症与氧化偶氮甲烷（AOM）和葡聚糖硫酸钠（DSS）