作者承认通过授予特蕾西J.羔羊儿童中心免疫与疫苗和美国国立卫生研究院新的创新奖（1DP2AI112242-01）的资金。作者还感谢纳塔利娅·P. Degtyareva为RAD1 S的慷慨捐助酵母。

1. UV诱变产生营养缺陷型酵母菌株<ol><li>生成生存曲线，以确定所需剂量的紫外线照射的<ol><li>根据标准方法26制备的YPD（酵母提取物蛋白胨葡萄糖）的媒体，在表1中列出的其它试剂和接种单个菌落到5-10毫升的YPD培养基。孵育在辊筒培养在30℃的CO / N至饱和至少8小时。 </li><li>通过稀释细胞1:10在水中的塑料比色杯中测定使用分光光度计O / N培养物的细胞浓度。稀释细胞在20ml无菌蒸馏水的10 7个细胞/ ml的浓度。 </li><li>倾稀释细胞进入无菌塑料培养皿，并与盖体取出，放置在板14厘米以下的UV灯泡。 </li><li>细胞暴露于串行5​​000μJ和10,000μJ剂量紫外线照射的，提取500微升细胞的每个增量，使得以下的细胞置身于0μJ，5000μJ10000μJ15000μJ20000μJ25000μJ30000μJ，4μJ，50000紫外线的照射μJ后进行采样。转移提取细胞样本无菌1.5毫升管，并在无菌水1:10的增量连续稀释。 </li><li>沉淀在在微量每个稀释离心细胞以16,000 xg离心1分钟。抽吸上清，重悬在无菌水中适当的电镀酵母细胞加入100μl体积。吸取全部体积悬浮细胞在含有YPD固体培养基平板，并使用无菌撒布在每个板对均匀分布的细胞。 </li><li>裹片边缘的封口膜，以防止媒体和盖板干燥铝箔，以防止光复活和紫外线诱发突变的修复。孵育板倒置，在30℃下2-4天以允许活酵母菌落（图1）的生长。 </li><li>算上numbe菌落R，可选择用钢笔的帮助下，划出菌落计数，手持电子计数器笔，或计数器站在放大倍率。积于UV照射的每个μJ总剂量接种的细胞的百分比，以产生用于照射酵母（图2）存活曲线。 注:单倍体酵母菌株可以预计需要较低剂量的照射相对于二倍体菌株紫外线到达相同百分比存活。酵母缺乏功能性DNA修复酶，如RAD1 S的应变酵母突变体，可以使用作为阳性对照，以指示紫外线照射的在非常低的剂量存在。 </li><li>确定紫外诱变的剂量通过参照建立在1.1.7生存曲线用于筛选。沿其中y等于50的存活曲线的点的x值是紫外线照射量在该酵母的50％存活。在这种低百分之存活筛选突变体可能会导致成功突变的菌株的较高产量，特别是对于二倍体酵母。的50％存活的剂量为WT S.布拉 ， 如图2中，是约18,000μJ。 注意:虽然这种高紫外线剂量增加突变的基因比感兴趣的营养缺陷型标记基因其他细胞通路的风险，该缺点必须针对需要以诱导突变的营养缺陷型标记基因的两个拷贝的平衡。对于单倍体菌株，其中仅一个基因拷贝必须进行突变，以更高的百分比存活筛选，例如在90％，降低的其他突变的风险，并仍允许足够产生营养缺陷型突变体。 </li></ol></li><li>紫外诱变和筛选营养缺陷型酵母菌株<ol><li>按照步骤1.1.1-1.1.3说明准备酵母。 </li><li>暴露酵母UV照射的对应于50％的存活率的剂量，如在1.1.8确定。对于WT S.酵母菌 ，这个剂量WA决心要为约18,000μJ（ 图2）。 </li><li>收集1毫升卷紫外线以16,000 xg离心在微量照射酵母和沉淀通过离心1分钟。吸去上清液和重悬的细胞在100μl无菌水中。 <ol><li>突变体的选择<ol><li>如果使用选择如与URA3 -营养缺陷型突变体，板照射酵母在含有5-氟乳清酸（5-FOA）的介质以选择缺少功能性的Ura3酶的细胞。 注:含有的Ura3的功能拷贝的任何酵母将5-FOA转换为毒素5-氟尿嘧啶，导致细胞死亡，并允许URA3的方便选择-即缺乏功能的Ura3 27菌落。类似的选择的方法是可能的LYS2和LYS5; TRP1;使用含有α氨基己二酸28媒体和MET2和MET15标记; 5-氟邻氨基苯甲酸29;和甲基汞30,31，分别。 </li><li>移液器100微升重新悬浮的细胞在含有5-FOA的基本培养基，并使用无菌吊具均匀涂覆板。包裹平板在封口膜孵育倒挂在30℃下2-4天以允许活酵母菌落的生长。 </li><li>通过restreaking到YPD，尿嘧啶出现在5-FOA平板的任何菌落表型- -确认URA3和5-FOA平板（图3）。用无菌牙签的尖端来收集单个菌落的一部分和整个新鲜的YPD，尿嘧啶轻轻拖动细胞-和5-FOA平板上。再次孵育包裹平板倒置，在30℃进行2-4天。 注:存活菌落将显示为凸起，大致呈圆形的生长而非存活细胞将仅显示为没有任何凸起的生长（图3）的不透明涂抹。 </li></ol></li><li>血肌酐突变体eening <ol><li>如果产生营养缺陷型突变的量选择方法都无法使用，制备紫外线的串行一点10稀释照射酵母细胞在无菌水中，并吸取稀释液到YPD培养基，用无菌吊具均匀涂覆板。 </li><li>裹在板封口膜，并培育倒挂在30℃2-4天。确定允许单个菌落生长，可以很容易地彼此区分，每板通常不超过约100个​​菌落其稀释。 </li><li>于本决定的稀释1.2.1-1.2.3和板细胞酵母说明样品重复紫外诱变。吸取稀释的酵母到YPD培养基中，使用无菌的吊具分发细胞，并孵育包裹平板倒置，在30℃进行2-4天。 </li><li>屏幕用于通过影印营养缺陷型到选择培养基缺乏感兴趣的代谢物。首先，安全无菌绒垫放在碟上的立场和反转用紫外线照射菌落板放置在绒布，标志着板的方向。 </li><li>接着，反转缺乏感兴趣的代谢物上的丝绒新鲜平板并轻轻地向下压，从绒转移细胞到板上。储存在4℃的原版。包装和孵化新盘倒挂在30℃2-4天。 </li><li>从YPD到选择性培养基通过重新裸奔殖民地替代影印，屏幕突变体。用无菌牙签的尖端来收集单个菌落的一部分，并轻轻地跨越新鲜的YPD板和缺乏兴趣代谢物的板拖动细胞。再次孵育包裹平板倒置，在30℃进行2-4天。 注意:必须小心选择单菌落和条纹出菌落多次确认真营养缺陷型细胞的同质群体。 </li></ol></li></ol></li><li>进一步证实照射细胞通过inoculat的表型ING单菌落到5-10毫升YPD双方，缺乏相应的代谢产物（如尿嘧啶-媒体的URA3 -突变体）的媒体。孵育在辊筒在30℃CO / N，以确认在​​YPD培养基中的细胞的生长，但不能在没有代谢物。 注意:虽然在固体培养基上生长模式应明确指出的酵母营养缺陷型状态，有可能对某些酵母耐受应力，并形成在固体培养基上小，缓慢生长的菌落，但尚未不能容忍在液体介质（未发表的观测值）相同的条件。接种到液体培养，因此应进行彻底检查照射紫外线的突变体的增长模式。 </li><li>为了证实营养缺陷突变体的长期存储，通过接种细胞准备甘油股票于10ml YPD，在30℃的辊筒O / N培养。离心沉淀细胞用于在2500 XG和吸媒体3分钟。悬浮细胞在50％的无菌过滤甘油，转移至冷冻管并储存于-80℃。 注:紫外线诱变酵母潜在地包含在比在感兴趣的营养缺陷型标记的其他多个基因突变。与验证使用营养缺陷型突变体在继续之前，这些菌株应进一步通过基因测序和pH值，胆汁酸胁迫性的评估，以及相关的益生菌菌株等特点进行了分析，如其他地方2描述。此外，使用PCR同源性或CRISPR的/ Cas9靶向更有选择性突变营养缺陷型标记应被视为替代紫外诱变4 - 6。 </li></ol></li></ol> 2.酵母转化<ol><li>酵母LiOAc转型<ol><li>接种单个酵母菌落入5-10毫升的YPD培养基中并在30℃的CO / N于辊筒孵育。 </li><li>以诱导对数期生长，提高质粒的摄取效率，德使用分光光度计termine细胞浓度测量在无菌水中1:10稀释的细胞在一个塑料杯中。稀O / N培养物至OD的0.16-0.2 600（约2×10 6 - 2.5×10 6个细胞/ ml）在50毫升新鲜温暖的YPD的孵育在轨道平台摇床设定为200转，直到培养细胞达到约1×10 7个细胞/ ml，通常为约4小时。 注意:转化效率可以为每质粒DNA微克成功转化的酵母细胞集落形成单位（CFU）的数目的函数来测量。每质粒DNA微克更多的转化菌落效率提高的结果。对数期生长过程中传代培养的酵母细胞和收集是增加转化效率12的一个因素。 </li><li>在2500 xg离心沉淀细胞离心3分钟。 </li><li>通过重悬佩尔吸出上清液并转移细胞到1.5 ml微量离心管等在1ml无菌水中。 </li><li>离心沉淀细胞在微量16000×g离心1分钟，吸出上清液，并再悬浮于1ml的TE / LiOAc液（Tris EDTA LiOAc）缓冲液洗涤细胞。 </li><li>重复离心和重悬细胞于TE / LiOAc缓冲至2×10 9细胞/ ml的浓度。 </li><li>制备转化混合物与每个如下:50微升的TE / LiOAc缓冲制备的酵母，5微升载体的DNA（10微克/微升）和1微升质粒DNA（1微克）的。质粒DNA的微克可滴定作为增加的DNA量可能会或可能不会导致转化效率32增加 注:对于缺少一个营养缺陷型标记的突变酵母菌株中，只有编码标记一个质粒可以每个样品进行改造。此外，利用编码易于检测的蛋白，如绿色荧光蛋白的质粒，将允许有效测定正确折叠和异源PR的表达通过转型后的酵母菌株otein。 </li><li>对于每个准备，加300微升PEG / LiOAc / TE和涡彻底的。完整的细胞与聚乙二醇的孵育为有效转化13是必不可少的。 </li><li>通过将离心管中以200rpm放置到一个轨道平台振荡器的烧杯中孵育在30℃下为在搅拌30分钟的准备。 </li><li>添加35微升DMSO中至每个反应和热休克细胞15分钟在42℃水浴中。虽然有冲突的DMSO中33的附加 ​​益处的报道，完整的酵母细胞的热休克已经显示出极大地提高转化效率12。 </li><li>通过经由离心造粒如2.1.5，抽吸或吸取掉上清，于1ml重悬浮无菌水洗涤细胞。吸管轻轻向上和向下，打破了细胞沉淀。 注:关键是要彻底去除上清因为媒体使用d。在产生的感受态细胞能抑制酵母菌的生长和集落形成。 </li><li>重复单元造粒如2.1.5，吸出上清液，并重新悬浮在100μl无菌水中的细胞。移液器全卷到选择性板使用无菌撒布均匀涂布板与转化酵母。 </li><li>裹涂布板的边缘在封口膜，以防止介质干燥，并培育倒挂在30℃下2天，以允许转化的酵母细胞的生长。成功，高效转化和酿酒酵母的营养缺陷型选择产生了大量的每转化制备菌落，虽然产率可以是用于其它菌株（图4）低得多。 </li><li>商店酵母板在短期内（一般为1-3周）倒过来，在4℃覆盖。作为长期储存在1.2.5中描述制备转化的酵母的甘油原液。 注:进一步的研究测试转化CFU的必要确定质粒稳定性和评估异源蛋白质的正确表达。质粒稳定性34，使用免疫印迹的透彻描述来检测从细胞样品17回收的变性蛋白，酶联免疫吸附试验（ELISA）来检测正确折叠三维蛋白16，并在酵母的研究中使用的GFP 35可用在别处。 图6显示了成功的GFP表达的转化的形象代表酵母相对于未转化的细胞。这种荧光蛋白的使用是有效地确定成功的异源蛋白的生产手段之一。 </li></ol></li><li>酵母的电穿孔<ol><li>接种单个酵母菌落入5-10毫升的YPD培养基中并在30℃的CO / N于辊筒孵育。 </li><li>使用分光光度计测量在无菌水中1:10稀释的细胞确定细胞浓度。迪琵琶O / N培养100毫升的新鲜温暖的YPD媒体的OD 600相当于约0.3。孵育在30℃下继代培养在轨道平台摇床设定为200rpm下，直到达到的OD 600约为1.6，通常是4-5小时。每次100ml亚文化将产生两个转化反应足够的条件细胞。 </li><li>在2500 xg离心沉淀细胞离心3分钟。吸出上清液并通过在50毫升冰冷的无菌水重悬洗涤细胞。通过制粒细胞，吸上清，在新鲜的50毫升冰水的冷无菌水重悬重复洗。 </li><li>再次沉淀细胞，并在50毫升冰冷的电穿孔缓冲液（1M山梨醇，1毫米氯化钙2）重悬。 </li><li>重复旋如2.2.3，吸去上清液，并悬浮细胞在20毫升的0.1M LiOAc / 10毫摩尔DTT。孵育细胞悬浮液上的辊筒在30℃下30分钟。在LiOAc和DTT细胞的预培养协同增加电穿孔36的效率。 </li><li>颗粒细胞作为2.2.3，上清，并在50毫升冰冷的电穿孔缓冲液中重悬洗。重复离心和重悬细胞的冰冷电穿孔缓冲液以1毫升的最终体积。 </li><li>制备在冰上:空调酵母细胞，无菌电试管，和质粒DNA。在1ml电穿孔缓冲液条件的细胞的最终再悬浮后，立即结合400μl的空调的酵母细胞用约1微克质粒DNA，并加入到冰冷的0.2微米的电穿孔杯中。的DNA量增加的使用可能会稍微提高转化效率11。在冰上孵育反应5分钟，然后用电穿孔集电穿孔至2.5千伏和25μF。 注:如2.1.7所述，缺少一个营养缺陷型标记的突变酵母菌株可以只用一个质粒编码每个采样突变标记物被转化。此外，使用编码易于检测的蛋白，如绿色荧光蛋白的质粒允许有效判定正确折叠和对变换随后的异源蛋白质的表达。 </li><li>传输电穿孔的细胞成8毫升1:YPD的1:1混合物:1M山梨醇和允许细胞在30℃下在辊筒孵育60分钟。 </li><li>丸细胞在2.2.3和在100μl1重悬:1 YPD:1M山梨醇。板全部体积在含1M山梨醇的选择性培养基，在封口膜包裹板的边缘，并孵育平板倒置，在30℃下2-5天以允许转化的酵母细胞的生长。 注:这是评价外源蛋白的正确表达，如2.1.14和2.2.7所述的试验转化酵母的关键。 </li></ol></li></ol> 3.转化酵母小鼠口服灌胃<ol><li>根据指南的实验室的管理和使用进行所有动物管理和处理程序imals和机构动物护理和使用委员会的批准。 </li><li>接种转化营养缺陷型酵母单菌落到5-10毫升的选择性培养基的制备O / N酵母培养物。在30℃下孵育培养物O / N为上辊筒的至少8小时，直到达到饱和。 注:质粒编码测试蛋白如GFP的使用将允许在管饲酵母易于蛋白表达的检测，如在4.7中描述。 </li><li>用于蛋白质表达的最大诱导和诱导对数期生长，从将O制备亚文化/ N培养物通过如2.1.2中所述稀释到OD 600当量为约0.16-0.2在50ml合适的介质中。 </li><li>确定传代培养细胞的浓度如在1.1.2和调整到10 9细胞/ ml。制备100μl的剂量为每只小鼠，每组几百微升额外体积，以提高准确度和缓解样品加载的。 </li><li>离心沉淀细胞在2500 XG为3分钟或在16,000 xg离心1分钟的离心。抽吸上清，重悬细胞，加入无菌水等体积和轻轻吹打上下。 </li><li>修正了一个合适的计灌胃针（15-20摹老鼠22克）到1毫升无菌注射器和负载酵母样品时，应确保消除任何气泡，并设置柱塞100微升增量。负载无菌水灌胃对照组小鼠的额外的注射器，并检查是否有任何污染酵母的存在。 </li><li>拾取到使用非优势手来管饲小鼠，用食指和拇指紧紧抓在脖子上（图6A）的皮肤。掖下的小手指的尾部，以防止下主体的运动。可以肯定的把手是安全的，并从以防止管饲期间内部组织损伤移动其头部禁止鼠标。 注:估计握住针对鼠标使得灌胃针多远应插入灯泡是即使在胸骨的剑突。插入这个长度针通常将允许灌胃针球进入胃。 </li><li>使用优势手，轻轻钓鱼沿着嘴和咽喉的背面的屋顶针，稍保持到中心的左边插入灌胃针入小鼠食道。等待鼠标吞针的灯泡和允许针稍微进一步下降到3.7（图6B）所估计的点。如果灌胃针插入过程中或如果鼠标在任何时候开始喘气感觉到任何阻力，轻轻取出针，并再次试图找到食道。 </li><li>鼠标已经吞噬了灌胃针的球后，轻踏注射器活塞管理酵母100微升（10 8 CFU）直接进入小鼠胃。 注:尽管小鼠不能管理18,19后呕吐任何灌胃解决方案</SUP&gt;，它有可能为管饲21,37期间发生回流。正确插入的管饲针，以及调节溶液的体积和粘度，可以有助于限制回流并确保准确的剂量。 </li><li>小心地从小鼠胃和食道灌胃针和鼠标返回到笼子里。检查小鼠呼吸，并且管饲后正常转动，以确保灌胃针被正确地在整个过程，并且没有溶液吸插入。 </li></ol> 4.鼠淋巴集结收获和隔离活酵母菌落<ol><li>在适当的时间点后灌服，一般是4小时，使用IACUC牺牲小鼠认可的方法。检查下一个脚趾捏缺乏响应能力，并使用一个辅助措施，如颈椎脱臼牺牲鼠标。另外的时间点，也可以进行测试，因为大量的研究已经表明，效率和启动定时采取跨越上皮颗粒依赖38,39。 </li><li>躺在充分暴露腹部鼠标和通过用70％乙醇喷雾消毒腹部区域。做一个横切口通过皮毛和皮肤用剪刀，小心不要破坏任何内部组织。手动撬切口打开进一步揭露腹膜，薄浆膜衬里覆盖腹部器官。轻轻提起腹膜，并作一横切口，显露肠子。 </li><li>小心使用钝钳逗小肠从肠系膜动脉，脂肪和其他组织的路程。从胃暴露小肠，在小鼠腹部的左上象限，盲肠，大袋肠组织中的大肠的开始。 </li><li>通过寻找1-3毫米沿着小肠不透明组织的大致圆形的补丁（图7）隔离淋巴集结。采用弧形夹层SCIS感器，切掉淋巴集结的圆顶，留下的利润，以确保没有周围的固有层被收集。 注:大多数小鼠有4-8轻松可见淋巴集结之间。与直接头顶照明的区域执行的程序将增加与淋巴集结可以可视化的难易程度。 </li><li>收集解剖淋巴集结在完整Iscove氏改性Dulbecco氏培养基（IMDM）。 注:这是为了防止酵母板胃肠道细菌污染使用无菌技术，包括抗生素在收集媒体的关键。 </li><li>通过一个40微米的细胞滤网过滤淋巴集结，以消除收集媒体。洗涤Peyer氏用新鲜的完全的IMDM修补过的50ml管中，并使用柱塞从1ml注射器轻轻打散淋巴集结。颗粒离心细胞紧张转速为1,800 rpm 7分钟。吸上清，重悬细胞大约100微升的最终体积。 </li><li>应用应变细胞上选择性酵母培养基，并使用板吊具均匀分布的细胞。裹片边缘在封口膜孵育平板倒置，在30℃下进行2天，以便用于从鼠淋巴集结中回收任何可行的酵母的生长。 注意:从淋巴集结"补丁酵母的恢复后需要进一步的研究，以确认该菌株能够提供正确折叠的异源蛋白质，以这些免疫组织。如在2.1.14中描述，这样的方法可包括免疫印迹，ELISA或荧光显微镜检测荧光蛋白如GFP 2,40。 </li></ol>

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作者什么都没有透露。

一起，这些协议本文描述所必需的开发和营养缺陷型益生菌酵母菌株的测试用于递送外源治疗性蛋白质的于肠的基本步骤。这种操纵和重组益生菌酵母的测试需要的技术和资源，与任何个人的实验室目前可能不熟悉。因此，尽管先前的许多研究已经说明上述协议多次酵母和小鼠品系，这些方法都没有作者的知识进行了详细，统一的形式提交。此外，本手稿特别强调适应当前标准化协议为益生菌酵母的基因操作，这是较差的特征在于比实验室常用的酵母菌株。两种诱变许多步骤和转化（第2部分）（部分1所讨论的），必须为这种二倍体的操纵，益生菌酵母isolat被优化上课。这份手稿还讨论了与动物处理（第三部分）和淋巴集结小肠的免疫组织（第四部分）的剥离相关的潜在缺陷。 由于许多工业和临床相关的酵母菌株不能立即适应大规模基因操作，它是生成菌株第一必要如可以种植和选择而没有昂贵的抗生素营养缺陷型突变体。紫外诱变就是这样的一个做法，允许营养缺陷型基因7.41快速非特异性突变。存活曲线可以容易地生成（图1和2），以确定用于筛选突变体的适当剂量。然而，这种方法具有引发断可能影响生长速率或酵母菌株的其它性能目标突变的风险。有针对性的基因敲除可代替使用PCR构建或CRISPR / Cas9系统生成。随后筛选或选择突变体（图3）允许营养缺陷型酵母的鉴定。通过电镀到5-FOA媒体使用的选择，例如，可以快速消除仍含有功能性URA3营养缺陷型基因的任何酵母。如果可能，该选择方法可以是优选的屏幕，这就要求产生的所有菌落分析。与任一选择或筛选，但是，它是执行个别酵母菌落的重复条纹到选择培养基，以确认营养缺陷型状态是至关重要的。 所产生的突变体的转化可通过不同的协议来实现。虽然LiOAc转化是有效的许多酵母菌株的转化，特别是为最常用的实验室S.酵母菌株 ，可替代的协议，如电穿孔可以转换其他酵母以更高的效率（图4）分离。每个新菌株应进行测试USI纳克多个协议，以确定用于转化的最佳条件。不同的孵育时间和DNA的浓度，例如，可以影响整体转化效率，应测试和优化每个菌株33。 口管饲法允许直接向鼠胃肠道，其免疫组织然后可以测定酵母和异源蛋白质的递送的受控剂量的这些重组酵母的。适当的口腔管饲法技术（图6）是至关重要的，以尽量减少动物的不适，并提高实验精度。此外，淋巴集结是评估从肠道重组酵母的摄取键部位。免疫组织这些簇抗原取样和黏膜免疫反应诱导的重要场所。大抗原，包括酵母3-6微米的直径，最有可能被淋巴集结的M细胞以越过ガ溶于strointestinal上皮细胞和免疫细胞相互作用。必须小心解剖淋巴集结时，以确保只有细胞从贴剂而非肠腔或固有层被收集（图7）内作出。还必须采取以下解剖评估在回收的酵母（图8）正确表达和异源蛋白质的功能的进一步步骤。从酵母裂解物和免疫印迹的总蛋白质的制备是，以评估蛋白质表达一个标准方法;然而，这种方法不能提供关于蛋白质折叠和功能的信息。为了评估蛋白质功能，酵母可以用质粒编码GFP的转化和从淋巴集结恢复后在荧光显微镜下进行分析，以评估官能GFP表达（图5）。 总之，这份手稿提出了一套统一的详细的实验方案跨越步骤来回回中号营养缺陷型突变体的产生益生菌酵母的来自鼠肠的恢复。通过编译不传统上落下专长的一个区域内的协议，这些描述将有助于进一步研究测试，以益生菌酵母设计为口服药物递送载体的免疫反应。作者希望这项研究将鼓励讨论和促进每个试验的酵母菌株的实验方法优化，实现最有效的方法新颖，益生菌基于重组疗法的发展铺平了道路。

益生菌微生物是有效和经济地提供异源蛋白对胃肠道一个有趣的电位的装置。这些微生物能够通过胃肠道存活通路还没有殖它1，使控制剂量和限制性的药物表示曝光的。此外，为了容易工程师这些生物体产生大规模的异源蛋白质的能力使它们的经济替代合成递送颗粒。然而，这样的方法的开发，作为使用益生菌酵母布拉酵母菌 2的营养缺陷型菌株最近证明，需要的实验室技术在给定研究中传统上不结合，从酵母和分子生物学到动物处理技术和免疫学方法的知识。因此，尽管本文所描述的各个过程本身并不新颖实验室协议，本手稿的目标是提出一种统一介绍所需益生菌酵母的实验测试作为药物递送载体的鼠胃肠道的技术。提供是为必要的协议汇编:1）代的酵母的营养缺陷型突变株，可以很容易进行遗传操纵的; 2）酵母培养的改造，表达外源蛋白; 3）重组酵母经口服灌胃肠道的管理;和4）从它们的异源蛋白质表达的鼠肠和评估可行重组益生菌酵母的回收​​。 首先，虽然用于酵母物种的操作存在许多阳性和阴性选择方法，阴性选择如通过利用营养缺陷型标记的增大的效率和缓解与酵母可转化和选择。使用抗生素转化的积极选择，在合作ntrast，显著增加酵母操纵的成本。此外，酵母在含有抗生素的固体培养基的选择可允许相对于合成滴出固体介质（未发表的观测值）的营养缺陷型酵母的选择未转化的背景菌落增加的生长。营养缺陷型酵母是缺乏酶的必需氨基酸或尿嘧啶的合成关键菌株。如果补充丢失的代谢产物或代谢基因，从而使消极的选择，当酵母接种到合成辍学媒体缺乏必要的代谢产物如酵母只能生长。许多常用的酿酒酵母的实验室菌株中实际上已经营养缺陷型突变体3。工业，临床和益生菌酵母菌株，但是，通常是原养与合成所需的所有营养物质的能力。为了使这样的酵母更高效的基因操作，营养缺陷型的基因可以被选择性地定位产生能够无抗生素选择菌株。 6 -营养缺陷型的标记基因特异性靶向可通过PCR介导的基因破坏依靠同源重组或最近通过CRISPR / Cas9目标4来实现。可替代地，紫外诱变可以快速生成，即使在酵母菌株为哪些变换与多个质粒在技术上是困难7营养缺陷型突变体。虽然PCR定位和CRISPR / Cas9已经被广泛地描述在其他地方，部分提出这个手稿之一是描述紫外诱变的方法来创建营养缺陷型菌株，将允许阴性选择，而不是酵母转化的积极的抗生素选择一个详细的协议。 在使用为异源蛋白质的口服递送这样的营养缺陷型菌株的下一个必要的步骤是酵母转化用质粒DNA。由于赞成票的第一个成功转型在1978年8报道了酿酒酵母牛逼的球状体，大量的修改进行了表征，以提高效率和易用性与酵母菌种可遗传修饰。利用电对DNA的成功转型为S.酵母最早是在1985年9节 ，至今已通过加入1M山梨醇培养渗透到细胞支持10提高。电效率此外，已显示依赖于酵母种和菌株，细胞数和生长的阶段，电体积，电场强度，和特定的缓冲区11。醋酸锂（LiOAc）变换，最初由Ito等人12所述，是最常用的变换协议之间，因为它不需要特殊的设备。附加分析表明，LiOAc酵母转化效率时，细胞在数中期收集大大增加生长并热在聚乙二醇（PEG）和DNA的存在下，在42℃12震惊。用PEG整个完整的酵母的培养是高效转化必需的，有可能通过提高DNA与细胞膜的附着以及通过在膜13等效果。锂本身也增加了完整细胞14的渗透性。尽管大多数实验室S.酵母菌株可以很容易地使用LiOAc变换3被转化，其它酵母物种可更有效地使用替代的协议变换。 巴斯德毕赤酵母 ，例如，是通过电穿孔，而不是LiOAc变换13最有效地转化，这是至关重要的，因此，要测试多个改造和方法，试图在基因修改未鉴定的酵母菌株，优化培养时间和试剂浓度。这份手稿从而描述了两种LiOAc TRansformation和电穿孔作为营养缺陷型突变体和野生型S的转化技术布拉 。感兴趣的读者可以直接用于酵母转化，替代协议和行动13,15的可能机制进一步讨论演变的深入描述最近的评论。用质粒编码易于检测的蛋白质的酵母的转化是为了确保正确的表达和异源蛋白质的功能下游测试进一步必要的。无数的不同蛋白质可以根据治疗研究的最终目的和通过免疫印迹，ELISA和其它技术可用于蛋白质检测的抗体来选择。对这些技术的协议已被彻底别处16,17描述，并且可用于通过比较标准曲线，以确定从转化的酵母异源蛋白质的生产水平。为目的的示范的，并显示出成功地生产在酵母生物学一个很常用的蛋白质，这手稿呈现质粒编码绿色荧光蛋白（GFP），其允许使用荧光显微镜后续检测转变。 到生产表达异源蛋白质是适当管理和胃肠组织内的这些微生物的检测，如在第三和第四部分中描述的益生菌微生物的同样重要。通过口服管饲法重组酵母的施用允许递送的酵母的控制量直接进入胃，从中C57BL / 6小鼠是自然不能呕吐18。然而，不当处理动物灌胃，并可能导致食管损伤，穿孔，胃穿孔，气管管理和吸入性肺炎19,20。可怜的技术和缺乏经验可以进一步提高小鼠的免疫反应和实验地区环境部门一道变异LTS，已经口服管饲法21,22归因于动物的压力。在适当的技术实践可因此不仅衰减动物的不适，但也可以增加的实验结果的精度。这份手稿介绍并演示了动物处理和口服灌胃的掌握剂量重组酵母的管​​理。 最后，这是至关重要的，通过分析淋巴组织用于酵母和异源蛋白质的存在下，以确认重组酵母的成功递送。可以最容易地且可预测地检测为酵母的存在下消化道免疫组织是淋巴集结。淋巴集结沿小肠二级淋巴器官是粘膜免疫应答的诱导23关键点。从内腔抗原通过微皱transcellularly转印在上皮（M）的细胞和被释放到淋巴集结，从而暴露括D抗原呈递细胞肠肠腔内容物。虽然在整个肠上皮粒子的摄取也可以由杯状细胞来实现，这些细胞已被证明仅占用颗粒小于0.02微米的直径24。跨上树突从CD103 +树突状细胞（DC）也采取了从肠道小颗粒管腔25延伸;然而，目前还没有报告表明CD103 +的DC占用比细菌大颗粒。因此，完好的益生菌酵母，3-6微米直径之间的平均大小，最有可能由M细胞吸收并转移到淋巴集结。这里描述的是用于收集和淋巴集结为可行的重组酵母的筛选的协议，虽然这过程也可以容易地适应用于评估益生菌摄取。 综上所述，评估重组益生菌酵母的交付rapeutic蛋白质在肠道需要实验室技术跨越分子生物学动物处理和免疫能力。 1这里提出是协议），它可以很容易地无抗生素负选择的生成和营养缺陷型酵母菌株的筛选，2）替代协议来转化酵母，并启用异源蛋白质的表达，3）的适当的动物处理方法和用于口服管饲法示范胃内递送重组酵母的，4）协议淋巴集结解剖和筛选可行的重组酵母和功能性的异源蛋白质。相结合，这些协议将允许产生和能够提供异源的治疗性蛋白的胃肠道的益生菌酵母株的测试。

<table border="0" cellpadding="0" cellspacing="0" width="704">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">SmartSpec 3000 Spectrophotometer</td>
			<td style="width:107px;">BioRad</td>
			<td style="width:107px;">170-2501</td>
			<td style="width:275px;">Example of spectrophotometer for determining cell concentration and OD600 of yeast cultures</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">New Brunswick Roller Drum</td>
			<td style="width:107px;">Eppendorf</td>
			<td style="width:107px;">M1053-4004</td>
			<td style="width:275px;">Example of roller drum for yeast culture incubation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UV Stratalinker 2400</td>
			<td style="width:107px;">Stratagene</td>
			<td style="width:107px;">400075-03</td>
			<td style="width:275px;">Example stratalinker</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Stuart Colony Counter SC6PLUS</td>
			<td style="width:107px;">11983044</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:275px;">Plate stand with magnification records colony count upon sensing pressure from pen</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Scienceware Colony Counter&#160;</td>
			<td style="width:107px;">F378620002</td>
			<td style="width:107px;">Bel-Art Scienceware</td>
			<td style="width:275px;">Hand held colony counter pen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Replica plating device</td>
			<td style="width:107px;">Fisherbrand</td>
			<td>09-718-1</td>
			<td rowspan="2" style="width:275px;">Example of replica plating stand and pads&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Velveteen squares</td>
			<td style="width:107px;">Fisherbrand</td>
			<td>09-718-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;L shaped sterile cell spreaders&#160;</td>
			<td style="width:107px;">Fisherbrand</td>
			<td style="width:107px;">14665230</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Deoxyribonucleic acid, single stranded from salmon testes</td>
			<td style="width:107px;">Sigma-Aldrich</td>
			<td style="width:107px;">D7656-1ML</td>
			<td style="width:275px;">Example carrier DNA for yeast LiOAc transformation</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Gavage needles</td>
			<td style="width:107px;">Braintree Scientific</td>
			<td style="width:107px;">N-PK&#160;002</td>
			<td style="width:275px;">For mice 15-20 g, the suggested needle is a 22 gauge (1.25 mm ball), 1 in long, straight reusable gavage needle. For mice weighing greater than 20 g, 20 gauge or larger straight or curved gavage needles may be used</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1mL sterile slip-tip disposable tuberculin syringe&#160;</td>
			<td style="width:107px;">Becton Dickinson</td>
			<td style="width:107px;">BD 309659</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Blunt forceps such as Electron Microscopy Sciences 7&#34; (178 mm) serrated tip, broad grip forceps</td>
			<td style="width:107px;">Electron Microscopy Sciences</td>
			<td style="width:107px;">77937-28</td>
			<td style="width:275px;">Example of blunt forceps needed for dissection&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Straight and curved dissection scissors&#160;</td>
			<td style="width:107px;">Electron Microscopy Sciences&#160;</td>
			<td style="width:107px;">72966-02 and 72966-03</td>
			<td style="width:275px;">Examples of scissors needed for dissection&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">IMDM</td>
			<td style="width:107px;">Life technologies</td>
			<td style="width:107px;">12440053</td>
			<td style="width:275px;"></td>
		</tr>
	</tbody>
</table>

transformation of probiotic yeast and their recovery from gastrointestinal immune tissues following oral gavage in mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

以下UV照射一个存活曲线的产生需要稀释的酵母细胞，使得不同的集落形成单位（CFU）能够形成的镀覆。收集上述各500μl的样品含有约5×10 6个细胞;然而，每板大于100个菌落都难以准确区分。电镀未稀释样品以及照射细胞的连续1点10稀释从而确保了CFU可以在每个UV剂量可以列举， 如在图1表明该CFU计数，乘以稀释因子，然后通过的总数量除以原始照射细胞，以确定在每个剂量存活百分比各500微升样品中，图2示出了二倍体野生型S的计算的百分比酵母菌细胞能够存活0μJ，5000μJ10000μJ15000μJ20000μJ，22,500μJ25000μJ35000μJ，50000μJ。这些数据确立了可用于查找对应于50％的存活率的剂量明确曲线。 UV剂量和酵母细胞的照射选择之后，它是屏幕突变菌落临界确认缺少的官能营养缺陷型标记基因。选择方法的使用，如在1.2.3.1描述并在图3中所示 ，显著增加的表型的确认的效率。示出的是由完整的Ura3利用5-FOA的转化的对毒素的5-FU 的URA3选择的一个例子。类似的方法可用于LYS2和LYS5; TRP1和 MET2和MET15，提高选择效率这些突变。必须注意的筛选过程中选择单个菌落。突变菌落在YPD和5-FOA，但不尿嘧啶的持续增长- ，板indicat上课营养缺陷型。 图4显示了野生型S.转化效率布拉 （SB）相对于实验室常用S.同时使用LiOAc（LiOAc）和电穿孔（电）技术酿酒酵母菌株 （SC）。虽然LiOAc转型是S.非常有效酵母转化效率为S.布拉使用电穿孔大大提高。 图5示出了使用荧光显微镜作为从转化的酵母分析正确蛋白表达的示例性方法。明（A）和荧光（B）图像示为S.酵母转化用URA3质粒编码绿色荧光蛋白，从转化的酵母展示异源蛋白质的功能表达。细胞可以固定使用涂在刀豆盖玻片更好的可视化（涂层5微升2毫克/毫升储液的水到每个22×22微米的盖玻片和空气干燥）。 图6A示出了C57BL / 6小鼠保持之前，为了经口管饲。手握鼠标牢牢使得鼠标无法头部在任何方向移动的背部和颈部。这保持允许灌胃针被准确地放置和组织损伤的风险降低。 图6B所示举行灌胃针鼠标燕子灌胃针后。在温育期间，处死小鼠的小肠中，应小心地从周围的组织梳理开， 如图7。这种操作允许容易识别的Peyer结和用于修补的干净的夹层而不收集任何周围的固有层。 最后 ，图8示出了从淋巴集结可行酵母CFU的典型的恢复。 </p&gt; <table fo:keep-together.within-page="1"><tbody><tr><td> 解决方案 </td><td> 酵母媒体和板 </td><td> 转型试剂 </td></tr><tr><td> 聚乙二醇（PEG）50％: </td><td> YPD: </td><td> TE / LiOAc: </td></tr><tr><td> 250克PEG 3350 </td><td> 20克蛋白胨</td><td> 50毫升10X TE </td></tr><tr><td> 500毫升无菌水</td><td> 20克葡萄糖</td><td> 50毫升10倍（1M）LiOAc </td></tr><tr><td>过滤消毒</td><td> 10g酵母提取物</td><td> 400毫升无菌水</td></tr><tr><td></td><td> 1升的水</td><td>过滤消毒</td></tr><tr><td></td><td>高压灭菌器</td><td></td></tr><tr><td> TE 10倍: </td><td> YPD平板: </td><td> PEG / TE / LiOAc: </td></tr><tr><td> 100毫米的Tris </td><td> 20克蛋白胨</td><td> 400毫升50％PEG </td></tr><tr><td> 10mM EDTA的</td><td> 20克葡萄糖</td><td> 50毫升10X TE </td></tr><tr><td> pH值至7.5，并过滤消毒</td><td> 20克琼脂</td><td> 50毫升10倍（1M）LiOAc </td></tr><tr><td></td><td> 10g酵母提取物</td><td></td></tr><tr><td></td><td> 1升的水</td><td></td></tr><tr><td></td><td>高压灭菌器</td><td></td></tr><tr><td> 20％葡萄糖: </td><td> 尿嘧啶-选择性培养基 </td><td> 载体DNA（SS DNA）: </td></tr><tr><td> 200克葡萄糖</td><td> 2克缺乏尿嘧啶的氨基酸组合</td><td>储存在-20℃，使用热为1-2分钟，在100℃熔融股线并存储在冰上之前</td></tr><tr><td> 1升的水</td><td> 6.7克酵母氮碱无氨基酸</td><td></td></tr><tr><td>过滤消毒</td><td> 1升的水</td><td></td></tr> &lt;TR&gt; <td></td><td>通过高压灭菌或无菌过滤消毒</td><td></td></tr><tr><td></td><td>在使用之前加入20％葡萄糖1:10 </td><td></td></tr><tr><td> 50％甘油: </td><td> 尿嘧啶-板: </td><td> 电穿孔缓冲: </td></tr><tr><td> 500毫升甘油</td><td>在250ml烧瓶中: </td><td>的1M山梨醇</td></tr><tr><td>水500毫升</td><td> 2克缺乏尿嘧啶的氨基酸组合</td><td> 1毫米氯化钙2 </td></tr><tr><td>高压灭菌器</td><td> 6.7克酵母氮碱无氨基酸</td><td>用蒸馏水填充</td></tr><tr><td></td><td> 150ml水中</td><td>高压灭菌器和储存在4℃ </td></tr><tr><td></td><td>在一个2升烧瓶中: </td><td></td></tr><tr><td></td><td> 20克琼脂</td><td></td></tr><tr><td></td><td> 750毫升水</td><td></td></tr><tR&gt; <td></td><td>高压釜烧瓶分开，然后用100毫升20％葡萄糖混合在一起</td><td></td></tr><tr><td> 完整的IMDM </td><td> 5-FOA +板: </td><td> LiOAc / DTT </td></tr><tr><td> 500毫升的Iscove氏改良的Dulbecco氏媒体</td><td>高压釜在2L烧瓶: </td><td> 0.1M的LiOAc </td></tr><tr><td> 5毫升青霉素链霉素谷氨酰胺100倍</td><td> 20克琼脂</td><td> 10毫摩尔DTT </td></tr><tr><td> 500微升2-巯基乙醇</td><td> 750毫升水</td><td></td></tr><tr><td> 10％热灭活胎牛血清</td><td>混合: </td><td></td></tr><tr><td> 2.5毫升丙酮酸钠100毫米</td><td> 6.7克酵母氮碱无氨基酸</td><td></td></tr><tr><td></td><td> 2克氨基酸组合没有尿嘧啶</td><td></td></tr><tr><td></td><td> 150毫升温水</td><td></td&gt; </tr><tr><td></td><td>当冷，加: </td><td></td></tr><tr><td></td><td> 0.05克尿嘧啶粉</td><td></td></tr><tr><td></td><td> 1克5-FOA </td><td></td></tr><tr><td></td><td>搅拌和过滤消毒</td><td></td></tr><tr><td></td><td>加入灭菌琼脂溶液</td><td></td></tr><tr><td></td><td>用100ml 20％葡萄糖混合</td><td></td></tr></tbody></table> 表1试剂列表。描述的是需要使每个用于本手稿的协议的解决方案，酵母媒介和板材，以及变换缓冲器的试剂。 <img alt="图1" src="/files/ftp_upload/53453/53453fig1.jpg" /> 图1.酵母的菌落生长在YPD介质。实施例的YPD板示出可行的集落形成的p单位（CFU）紫外线照射后robiotic酵母。细胞连续稀释，使得个人CFU可以区分并计数。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53453/53453fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53453/53453fig2.jpg" /> 图2.生存曲线二倍体益生菌酵母。可行S.数布拉 CFU占总铺板的细胞的百分比作图对于UV照射（实线）的每个μJ剂量。垂直红线表示μJUV剂量对应于该酵母菌株的50％的存活率。一个RAD1酿酒酵母突变体，它不能修复从紫外诱变破坏，显示为控制（虚线）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53453/53453fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。&lt;</a>/ A&gt; <img alt="图3" src="/files/ftp_upload/53453/53453fig3.jpg" /> 图3. URA3 的确认 - 紫外线的表型照射细胞在YPD，尿嘧啶 - 和5-FOA平板从个别紫外线突变体菌落的细胞使用无菌牙签的尖端收集并轻轻划过YPD，尿嘧啶挑染- ，和5 -FOA板。细胞首先在两个垂直交叉线划线，然后用一个新的牙签穿过第二线，并继续直到单个细胞分离扩频细胞。一个真正的ura3 -突变体（MUT）生长在YPD介质和在5-FOA存在下，但不是在不存在尿嘧啶的。控制URA3 - S. 酵母 （URA3 - ）和 URA3 + </<html> SUP&gt; S.布拉 （URA3 +）显示进行比较，并确认酵母培养基的适当准备。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53453/53453fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53453/53453fig4.jpg" /> 图4.转型 酵母属菌株 的效率 。野生型S.布拉 （Sb）和一个实验室S.酵母菌株 （ 钪 ）中使用所描述的LiOAc（LiOAc）和电穿孔（电）协议变换。结果被绘制为均值CFU每编码卡那霉素抗性标记的质粒微克获得。条表示重复实验用误差棒描绘平均值的标准误差的平均值。ES / ftp_upload / 53453 / 53453fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图5" src="/files/ftp_upload/53453/53453fig5.jpg" /> 通过转化酵母图5.功能性蛋白表达。S.使用荧光显微镜酵母空质粒（A）和质粒编码GFP（B）的转化进行分析。荧光绿色荧光蛋白基因质粒转化酵母细胞表示成功生产功能性绿色荧光蛋白。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53453/53453fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/53453/53453fig6.jpg" /> 图6.正确处理一个C57BL / 6小鼠口服灌胃，鼠标是举行tightlý在非优势手用下的小手指夹着尾巴使得没有运动是可能的（A）中 。灌胃针插入沿口的屋顶咽。鼠标允许吞下灌胃针的灯泡，使溶液然后进入胃的活塞被压下（B）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53453/53453fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/53453/53453fig7.jpg" /> 图7.准备和解剖Peyer氏补丁。小肠被示为与其他内部器官和组织解剖了，用箭头指向几个集合淋巴小结的补丁。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53453/53453fig7large.jpg" target="_blank">请点击这里查看大VERSI</a>这个数字的。 <img alt="图8" src="/files/ftp_upload/53453/53453fig8.jpg" /> 图8.从Peyer氏补丁酵母回收。从与 S管饲小鼠后解剖，均质检测存活的CFU，和总淋巴集结细胞的电镀的一个例子布拉 。细胞铺到YPD酵母培养基，并在30℃培养2天。 CFU每鼠收回的典型产量小于10 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53453/53453fig8large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice at JoVE.com

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

这里介绍的是，可以用于开发，变换，管理，和测试益生菌酵母布拉酵母菌中异源蛋白质表达技术的统一描述。

益生菌酵母的转型及其胃肠道粘膜免疫组织恢复继小鼠口服灌胃

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

transformation-probiotic-yeast-their-recovery-from-gastrointestinal

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

12.3K 次观看.  Emory University School of Medicine. 这里介绍的是，可以用于开发，变换，管理，和测试益生菌酵母布拉酵母菌中异源蛋白质表达技术的统一描述。 

视频: Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

科研

免疫与感染

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

测量细菌感染小鼠和免疫反应李斯特菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

口头传播的<em&gt;单核细胞增生李斯特氏菌</em小鼠通过食入被污染的食物

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

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 ...

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

原位检测使用地高辛标记的DNA探针石蜡包埋组织中的细菌靶向16S rRNA基因

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

分离，鉴定和牙龈免疫细胞网络的功能性考试

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

在免疫细胞在不同组织与疾病相关的分布和小鼠实验评价性自身免疫性脑脊髓炎的诱导

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data have encouraged pulmonary tuberculosis vaccines as a promising strategy to prevent pulmonary disease, which is responsible for transmission. In this work, we describe the methodology used to demonstrate in the mouse model the benefits of intranasal BCG vaccination when compared to subcutaneous. Our data revealed greater protective efficacy following intranasal BCG administration. In addition, our results indicate that pulmonary vaccination triggers a higher immune response in lungs, including Th1 and Th17 responses, as well as an increase of immunoglobulin A (IgA) concentration in respiratory airways. Our data show correlation between protective efficacy and the presence of IL17-producing cells in lungs post-Mycobacterium tuberculosis challenge, suggesting a role for this cytokine in the protective response conferred by pulmonary vaccination. Finally, we detail the global workflow we have developed to study respiratory vaccination in the mouse model, which could be extrapolated to other tuberculosis vaccines, apart from BCG, targeting the mucosal response or other pulmonary routes of administration such as the intratracheal or aerosol.

We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

保护疗效及肺部免疫反应皮下和鼻内BCG管理在小鼠

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

Subcellular Fractionation from Fresh and Frozen Gastrointestinal Specimens

The purpose of this protocol is to fractionate human intestinal tissue obtained by endoscopy into nuclear and cytoplasmic compartments for the localization analysis of specific proteins or protein complexes in different tissue states (i.e., healthy vs. disease). This method is useful for the fractionation of both fresh and frozen intestinal tissue samples; it is easily accessible for all laboratories and not time consuming.

Here, we present a protocol to perform a simple cellular fractionation for the subcellular separation of cytoplasmic and nuclear proteins in human fresh and frozen intestinal biopsies.

新鲜冷冻胃肠标本的亚细胞分离

The purpose of this protocol is to fractionate human intestinal tissue obtained by endoscopy into nuclear and cytoplasmic compartments for the ...

Probiotic Studies in Neonatal Mice Using Gavage

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult mouse models to neonatal diseases is limited. To better understand disease progression, host responses and long-term impact of interventions in neonates, a neonatal mouse model likely is a better fit. The sparse use of neonatal mouse models can in part be attributed to the technical difficulties of working with these small animals. A neonatal mouse model was developed to determine the effects of probiotic administration in early life and to specifically assess the ability to establish colonization in the newborn mouse intestinal tract. Specifically, to assess probiotic colonization in the neonatal mouse, Lactobacillus plantarum (LP) was delivered directly into the neonatal mouse gastrointestinal tract. To this end, LP was administered to mice by feeding through intra-esophageal (IE) gavage. A highly reproducible method was developed to standardize the process of IE gavage that allows an accurate administration of probiotic dosages while minimizing trauma, an aspect particularly important given the fragility of newborn mice. Limitations of this process include possibilities of esophageal irritation or damage and aspiration if gavaged incorrectly. This approach represents an improvement on current practices because IE gavage into the distal esophagus reduces the chances of aspiration. Following gavage, the colonization profile of the probiotic was traced using quantitative polymerase chain reaction (qPCR) of the extracted intestinal DNA with LP specific primers. Different litter settings and cage management techniques were used to assess the potential for colonization-spread. The protocol details the intricacies of IE neonatal mouse gavage and subsequent colonization quantification with LP.

This study details the process of gavaging precise amounts of probiotics to neonatal mice. The experimental set-up was optimized to include but is not limited to probiotic dosing, methods of administration, and quantification of bacteria in the intestines.

新生儿小鼠益生菌的研究

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult ...

益生菌酵母的转型及其胃肠道粘膜免疫组织恢复继小鼠口服灌胃

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