我们要感谢大卫·R·罗德里格斯，马的平面设计和摄影在这个手稿。这项工作是由来自GCCRI，美国国立卫生研究院/国家行政学院（5R21AG033339）和癌症中心支援津贴（P30CA054174）到UTHSCSA流式细胞仪核心设施和UTHSCSA先进的核酸核心设施提供资金支持。

从LacI的转基因小鼠在C57BL / 6背景白细胞不表达的Sca-1（ 图1）。因此，如果的Sca-1是用于细胞纯化的标记物，这些小鼠中需要用合适的菌株获得的Sca-1的表达被划线，在这种协议定期的C57BL / 6（B6）小鼠和LacI上的杂交的F1 （C57BL / 6）的转基因小鼠（LacI的 ）是使用（ 图1）。在这个协议中使用的细胞群，LSKs和中医师表示最小种群骨髓。为了从唯一的后腿，或从至少四个收获小鼠骨髓时，净化至少2×10 5每个/排序，从大约十相结合的小鼠骨髓的时候也臀，前肢-，脊柱骨，和胸骨被使用。 使从骨髓和脾的单细胞悬浮液。骨髓中的一个小部分被用作是，大部分用于PUR通过荧光激活细胞分选（FACS）IFY LSKs和分化的骨髓祖细胞， 即中医和粒细胞/单核细胞祖细胞（总纲图）。骨髓细胞的分离和流式细胞仪净化这些人群的其他地方30-32描述。约六个独立的各种需要，以确定种群间基因突变的频率显著差异。 下面的协议，用于测量自发体内突变频率中纯化造血亚群体适于从几个Stratagene公司的说明书33-35基于来自Kohler 等 18原来工作的现有协议33-35和这个协议之间最重要的区别，用相对较小的数字的单元格时，必需的，包括试剂的体积差异和时间，用于蛋白酶K的孵化温度。 收集所需的细胞群，分装细胞的1-ml离心管后（每等分细胞数， 见表1）。离心机在266×g离心7分钟，在4℃下小心吸出上清液。把管放入液氮中5分钟，然后将它们传输到-80℃冷冻以供进一步使用。样品可以贮存至少6个月。 2。基因组DNA的分离<ol><li>取样品从-80℃冰箱。加入500μl冰冷的DNA的裂解缓冲液（ 表3）的样品管中。涡流管3-5秒中速，并将管置于冰上10分钟。 </li><li>离心管12分钟，4,000×g离心，4°C。小心丢弃〜450μL上清液。降速为3-5秒的管。使用玻璃毛细管小心取出上清液的剩余部分。风干吨的内壁他管，直到没有液滴是可见了（〜2分钟）。 </li><li>加入2μl的RNace，它核糖核酸鸡尾酒和10微升的1 M DTT至100微升的DNA消化缓冲液（ 表3）。根据将要处理的样本的数目调整音量; 20微升该DNA消化溶液的每个样本是必要的。加入20微升的这种以细胞沉淀后，尽量让小球从底部脱离本身，轻轻敲击管子用手指或笔。 注意：可能很难看到，因为低细胞数目的颗粒。 </li><li>加入20微升蛋白酶K溶液*（ 表3）每个样品，轻轻地再次轻按管。 *注意：该蛋白酶K溶液应预热在50℃的水浴，2分钟，以激活酶后才能使用。 </li><li>在50℃水浴中立即放置管。根据该指引，消解样品<strong&gt;表1。点选管轻轻地每10分钟。 注意：对于这种小数量的细胞，该水浴的温度与消化时间是成功地获得高品质的基因组DNA的绝对关键的。 </li><li>制备的DNA的透析系统在冷室（ 图2）。倾600毫升TE缓冲液中（ 表3）放入600ml玻璃烧杯中，加入一个小的磁力搅拌棒，以使缓冲器可以透析过程中进行搅拌，并让（0.025毫米孔径）膜浮在缓冲器的表面上。每个样品一膜; 1-4膜可以在一个单一的透析烧杯中。就用剪刀识别各样品的膜的边缘的标记。 </li><li>经过适当的消化时间，增加现在非常粘稠的基因组DNA *小心到浮动膜片的中心（ 图2）。盖上烧杯立即用铝箔。透析的基因组DNA在4℃〜16-20小时，搅拌缓冲平缓。 *注：当粘性DNA溶液时，使用移液器吸头与宽的开口。 </li><li>第二天倒600毫升新鲜配制的TE缓冲液（ 表3），以一个干净的玻璃烧杯，膜，用勺子非常仔细地转移到新的烧杯中，盖上铝箔的烧杯中。透析另外2小时。 </li><li>从与用勺子的TE缓冲液的烧杯中取出透析膜并转移DNA溶液到新的无菌1毫升管只有最粘的“丛”。样品保存在4°C。继续诱变试验，第二天或最多1.5个月后。对于纯化的人群，等待至少1周。 </li></ol> 3。对大肠杆菌制备的托盘大肠杆菌 /噬菌体文化（第1天） 每个托盘将包含两个不同的层;琼脂层的底部和琼脂糖层在包含X-gal的顶部。 大肠杆菌大肠杆菌/噬菌体溶液将被加入到后者。细胞中分离出来的数量和类型将决定有多少塔板是必需的。参考表1，计算所需的托盘和这部分协议的解决方案，随后的量数。下面将产生〜60塔板，其中一个有经验的人可以很容易地处理。 <ol><li>准备以下介质： <ol><li>用于底层，制备6×2升烧瓶，每个含有1600毫升双蒸2 O。添加NZY粉（21克/ L）和琼脂（15克/升）;拌匀。 </li><li>用于顶层，制备4×1升烧瓶，每个含有800毫升双蒸2 O。添加NZY粉（21克/ L）和琼脂糖（7.2克/升）;拌匀。 </li><li>为培养大肠杆菌大肠杆菌，加2.5克NZY粉末的每个2×250毫升烧瓶中，并把体积至100ml双蒸2 O; </li><li>对于在步骤8中使用的确认托盘，添加8.4克NZY粉和6克的AGAr以1升的烧瓶中，使体积为400毫升双蒸2 O。 </li></ol></li><li>盖上铝箔烧瓶开幕。搅拌均匀，高压灭菌121℃，15磅30分钟。 </li><li>从高压釜中取出烧瓶（非常炎热！）。小心摇动烧瓶混合琼脂和琼脂糖，然后将它们放置在50℃水浴。当水浴已达到50℃再次，从2升瓶（步骤3.1.1）倒入约150ml NZY琼脂在每个托盘（底层）。 </li><li>使琼脂固化在室温下至少2小时，然后反转，打开的托盘。放置在托盘的底部上的盖子，45°偏离中心（ 图3），并让干燥30分钟。合上纸盘和离开O / N在室温。 </li><li>同时，准备SCS-8 E.大肠杆菌培养的第二天。从两个250ml烧瓶的1 *（步骤3.1.3），取5毫升NZY培养基（ 表3），并转移到一个无菌的14米升管。补充与62.5微升麦芽糖/ 硫酸镁溶液中，加入10微升的SCS-8 E的大肠杆菌甘油。在37℃，在250-300转，而摇晃3-4小时。使用15微升这种文化的接种95毫升NZY肉汤（在250毫升手枪瓶），文化O / N在37℃的摇床（250转）。 *注：其他的250毫升烧瓶中，可用于以后如果需要的话，调整培养物的OD（步骤4.1） </li><li>取1升量瓶中，用琼脂（步骤3.1.4），倒〜6-7毫升NZY琼脂60 mm培养皿（这将是足够的〜50的菜肴，所需要的步骤8）。让琼脂变硬（〜10分钟），然后反转并用塑料。这些菜可以保持在4℃最多一个月。 </li></ol> 4。对大肠杆菌制备的托盘大肠杆菌 /噬菌体文化（2天） <ol><li>检查SCS-8 E的外径大肠杆菌培养（步骤3.5）的分光光度计。调整外径600 0.6用NZY培养基（ 表3）（步骤3.1.3），然后将烧瓶置于冰上停止增长并保持在冰上，直到准备使用。这将用于步骤6.3和8.2。这种文化可以保存5天，在4°C。 </li><li>空气干燥所有检测托盘（倒前一天）为〜5小时（ 图3）。 </li></ol> 5。包装基因组DNA（2天;续） <ol><li>拿橙TRANSPACK所需数量的离心管，出-80℃冷冻并把他们放在干冰，直到准备使用;需要一个橙色TRANSPACK管要执行的每个包装反应。适当地标记每个管。有基因组DNA样品（步骤2.9）准备上冰。 </li><li>这一步应该执行一管的时候。完成移动到下一个DNA样本前完成步骤。解冻一根橙色管* 注1快速：用你的手指，直到大部分被解冻，然后把在冰上。采取相应的基因组DNA样品，并立即8-12微升样品* 注2,3转移到橙色管。通过轻轻拍打管，用手指轻轻地上下吹打3倍，以及混合的内容。尽量不要混合时产生气泡。将管置于30℃水浴中90分钟。 *注1：一个快速旋转的离心可能是必要的，以收集从内壁的所有内容和盖。 *注2：添加样品的体积取决于用来产生该样品的细胞数：11-12微升从2.0-5.0×10 5个细胞制备的样品，10微升的1.0×10 6个细胞的样品，和8微升为1.5×10 6个细胞样品。 *注3：DNA仍然是非常粘稠的，采取的DNA出的吸管尖推至样品管的底部，小心地拧绕的前端靠管的内壁。 </li><li>取1或2个蓝色TRANSPACK TUBES *在-80℃冷冻并把他们放在干冰，直到进一步使用。解冻他们迅速和12微升转移到每一个橙色的管子。轻轻吹打向上和向下3次混合的解决方案。旋管下2-3秒，然后点选管，用手指作进一步混合，并立即返回到30℃水浴中另外90分钟。 *注：使用1蓝管5-6反应和2个蓝色管10-12的反应。 </li><li> 90分钟后，加入稀释用970微升SM缓冲液*（ 见表3）每个反应，使反应停止，并涡旋以中速，持续5秒。把试管置于冰上，直到进一步使用。 *注：如果细胞数是≤5×10 5个，则使用500微升SM缓冲液（ 表3），以使反应停止; 2管（同一样品的）然后可以稍后组合（步骤6.4）。 </li></ol> 6。电镀包装基因组DNA（第2天;续） <ol><li>关闭倒琼脂吨开设了干燥，早上的光线。标签的托盘每个样品。 </li><li>溶于4.8克X-gal的16.8毫升N，N-二甲基甲酰胺（所需的步骤6.5）。立即搅拌均匀，放在摇床平台。防止光。溶液应澄清在20-30分钟。 </li><li>分装SCS-8 E.大肠杆菌细胞。对于每一组的托盘*，使用一个50ml锥形管中。标签的管与被包装的DNA样品的名称。加入2ml E的菌悬液为每个纸盘。 *注：例如3×10 5的绿化总纲图需要一套8盘（ 见表1）。因此，两等份，需要8×2 = 16盘。保持等份分开，从而准备两份50ml试管，每8×2 =16毫升E。菌悬液。 </li><li>加入1毫升包装的DNA样品*（步骤5.4），以适当的50ml管中含有SCS-8 E。大肠杆菌等分拌匀。这孵育E.用颤抖的衍丰大肠杆菌 /噬菌体混合乌兰巴托（250-300转），在37℃下23分钟。 *注意：如果DNA是从≤5×10 5个细胞中提取，将500μl的SM缓冲液，使反应停止（步骤5.4）。在这个步骤中，管可以组合并添加到1 50ml锥形管中。 </li><li>当E。大肠杆菌 /噬菌体混合物孵化，开始筹备顶层琼脂糖层。加入5 ml X-gal的/ N，N-二甲基甲酰胺溶液（步骤6.2），以每800毫升烧瓶的顶层琼脂糖溶液（来自步骤3.1.2;保持在50℃）。的X-gal的最终浓度为1.5毫克/毫升。当加入到琼脂糖的X-gal的可能沉淀一点。漩涡溶解在X-gal和把瓶子放回50℃水浴。 </li><li>就拿E.大肠杆菌 /噬菌体混合出的孵化器（步骤6.4）。每个样本需要多个托盘，每个托盘，需要50毫升X-gal/agarose解决方案（步骤6.5）。倾X-gal/agarose所需的体积为每个样品（ 即 50毫升×在托盘的数量）较大的无菌塑料瓶，并添加适当的大肠杆菌大肠杆菌 /噬菌体混合。摇动瓶子混合。在45-50毫升50毫升锥形管等份分割混合物。管的数量应该是相同的要求为该样品托盘的数量。 注意：吃剩的X-gal/agarose解决方案将在步​​骤8.3中使用。该溶液可以被存储在一个50℃的水浴中，直到进一步使用。 </li><li>倒在底部的检测托盘的一半容量为50毫升顶层琼脂糖混合物。由稍微倾斜试验托盘在一个方向迅速蔓延的琼脂糖。 注：琼脂糖将降温非常迅速，将成为不可能分布在托盘。因此，此步骤需要执行相对快速和琼脂糖是一直保持在50℃。 </li><li>允许顶层琼脂糖硬化至少15分钟。然后，反转并打开检测盘，让他们风干30分钟（ 图3）。 </li&gt; <li>合上托盘，孵化倒立（顶部底部琼脂层侧）的检测盘在37℃下15-16小时。不要堆叠超过5盘。 </li></ol> 7。一个推定的突变频率的测定（3天） <ol><li>从37℃培养箱中取出托盘，让他们冷静下来。算上半透明斑形成单位（PFU）每个托盘上，随机选择托盘上的2个部位，绘制正方形* 2.5×2.5厘米2或5×5厘米2，并计算所有个PFU在每平方米有标记细胞计数器（ 图4A，B）。 *注：为了绘制方使用的装置， 如图5。如果计数的个PFU的数量≥40，可使用更小的正方形，否则使用较大。 </li><li>利用计算的平方的平均值，由96如果计算具有较小的平方乘以;或24，如果计数与大的一个乘以这个数。加个PFU数计算所有纸盘从SA我品尝。这是个PFU的该样品所产生的总数量。 </li><li>接着，计数在每一个托盘上的突变斑块。现在的托盘已经冷却下来，这将有助于找出突变斑块。为了进一步方便验出蓝色斑块，移动托盘在红色和/或白色表面，红色和白色的纸张很好地工作。圆圈上的任何蓝斑用标记笔。记录每一个突变体的蓝色的形状和强度（ 例如全，圆形，扇形， 图4C）36,37。 </li><li>通过个PFU的总数 （步骤7.2）将突变个PFU的数量（步骤7.3）确定推定的突变频率对于每个样品。 </li></ol> 8。假定的突变体斑块（3-5天）的验证<ol><li>使用巴斯德吸管，芯各突变体（蓝色）PFU和插头转移到250微升无菌的SM缓冲液（ 表3）。加入25μL氯仿，涡旋，持续5秒在4°C D店继续次日或下一个步骤继续之前离开2小时在室温，以验证该突变PFU确实是一个突变。样品可以储存在4℃下至少1年。 </li><li>标签酮1 ml和1个4毫升的无菌管中插入的每个突变。在新1毫升管，稀释从琼脂塞在无菌的SM缓冲液（步骤8.1）1:50（2微升样品放入100微升SM缓冲液（ 表3）），涡旋简要，预留释放的噬菌体悬浮。到4毫升的无菌试管中加入200μl的SCS-8 E.大肠杆菌培养物（来自步骤4.1）和2μl从相应的1毫升管中稀释的噬菌体，在37℃下进行5-10分钟。 </li><li>加2.5毫升含有X-gal的（从左以上从步骤6.6），以每次4毫升管，旋流管混合后，倾注在60毫米NZY琼脂平板上的顶层琼脂糖的预先倾（步骤3.6）。离开10分钟（以令t他最琼脂糖凝固），反转菜和孵化O / N为37°C。 </li><li>第二天早上，从孵化器中取出培养皿，并确定蓝色个PFU对每道菜（ 图6）的比例。当个PFU的70％以上是蓝色的，一个突变被认为是证实[38,39]。芯从盘的突变体斑块，放入1.5ml的螺丝顶管，含有250微升SM缓冲液（ 表3）和25微升氯仿，它现在已准备好用于测序。 </li><li>当50％或更少的PFU的是蓝色的，它不被认为是真正的突变体38,39。当蓝色个PFU的频率在60-70％之间，回来检查，在对斑块的形状的说明;如果PFU的形状是“全”，进行测序。 </li></ol> 9。测序在LacI的Ğ烯突变<ol><li>建立PCR反应在25μl反应体系，其中包括1.5μ;升斑块上清液（模板，步骤8.4）中，正向引物SF1（5&#39;-GGAAACGCCTGGTATCTT-3&#39;）和反向引物SR2（5&#39;-GCCAGTGAATCCGTAATCA-3&#39;）。根据制造商的说明书使用PCR扩Taq聚合酶试剂盒。循环条件如下：94℃2分钟，然后35个循环的94℃20秒，60℃20秒，72℃2分钟，随后72℃5分钟的最后一个步骤。 </li><li>取各反应的5微升的等分试样，并运行在0.8％琼脂糖凝胶上，以确认扩增。 </li><li>清理根据制造商的说明使用Augencourt净化试剂盒的PCR反应。 </li><li>定量模板DNA。 </li><li>使用〜100ng的PCR产物作为模板DNA进行测序。在使用相同的PCR引物作为测序引物（见上文）的两个方向的序列的扩增子。 </li><li>装配序列，并与LacI的参考序列40对齐，以检测穆塔蒂附件。 </li></ol>

作者宣称没有竞争的财务权益。

本文中描述的体内诱变法是基于最初由Kohler 等人，18该模型利用λ噬菌体载体携带的lacI报告基因产生的LacI的转基因小鼠模型。这两个COS网站侧翼矢量允许一个相对简单的恢复和后续包装成感染性噬菌体颗粒，用来感染大肠杆菌大肠杆菌 。一个蓝色的牌匾将被噬菌体感染的大肠杆菌产生含有突变基因LacI的大肠杆菌 。蓝色的牌匾是对一?...

在大多数组织中，分化的细胞具有有限的寿命。为了保持功能完整，长寿命，组织特异性干细胞不断产生祖细胞，这反过来会产生所需的特定的组织的功能的完全分化的细胞。干细胞还可以通过一个叫做自我更新过程中补充自己的车厢。因此，干细胞是负责维护他们居住。因此组织的功能完整，是非常重要的，他们都配备了强大的机制来检测和潜在的修复受损的DNA。如果没有，他们可能获得多个基因（可能有害）的扰动，可以通过他们的后代继承。了解在一个生物体的寿命怎么干细胞的安全，保卫自己的基因组是一个重要的问题，可以帮助我们理解为什么基因组不稳定性与癌症和其他一些与年龄有关的疾病（在1,2审查）相连。 3-6测量某些类型的DNA修复。我们发现，例如，在造血系统中，双链DNA的断裂，可通过同源重组（HR）或非同源末端连接（NHEJ），后者是较低保真度的修复过程，从而使增加的风险修错误。两者都被利用在造血干细胞（HSCs）4,5，然而，在小鼠中，似乎它主要是NHEJ在造血干细胞，而早期祖细胞利用HR 4。类似的观察是为干细胞在皮肤6。有趣的是，在人类造血干细胞的人力资源，而不是NHEJ，似乎是首选的双链断裂3的修复机制。无论这两个物种之间的功能差异是真实的还是仅仅代表一个技术或实验的差异，还有待观察。 干细胞的剧目，以修复受损的DNA很可能包括其它DNA修复机制，如碱基切除修复（BER），核苷酸切除修复（NER）和错配修复（MMR）。 BER和NER负责修理单个或多个碱基对病变的单链DNA，而MMR修复碱基 - 碱基错配和插入/缺失环;这些类型的DNA损伤不能被NHEJ或HR修复。支持这一概念是从造血系统证明在这些途径和异常在HSC室7-9中的一个变化之间的联系，以及显影骨髓增生异常综合征10-16，一种疾病，起源于风险增加若干研究HSC和与增加基因组不稳定性随着病情的发展17有关。到目前为止，还没有报道的误码率，净入学率，和MMR直接在造血干细胞的测量。 除了 ​​阐明了在一个机械控制电平组织完整性的各种处理，就必须能够测量突变的DNA的程度，从而使像差在这些过程中的一个后果可以进行测试， 例如 ，在正常与基因改造的干细胞或老与年轻。然而，有关测定的发展是因为组织特异性干细胞的缺乏及缺乏的培养条件，可以保留“干细胞”的困难。此外，这样的测定应易于进行环境和遗传操作。一种可能的解决方案，这些限制和要求是采用了专门设计的，以检测DNA突变的小鼠模型。 MUL已经开发tiple转基因小鼠模型进行突变检测。例如，LacI的转基因小鼠18携带一个可恢复的λ噬菌体载体编码了LacI报道系统，其中的了LacI基因编码Lac操纵的抑制剂和作为突变记者。待了LacI基因的突变，lac 操纵子被激活，并且β-半乳糖苷酶的生产。 β-半乳糖苷酶裂解产色底物X-gal的（5 - 溴-4 - 氯-E-吲哚基-β-D-吡喃半乳糖苷），它把它的蓝色。该COS的网站侧翼LacI的载体可以轻松地恢复由λ噬菌体的蛋白质和E的后续感染大肠杆菌 。培养大肠杆菌感染后， 大肠杆菌琼脂糖含有X-gal的基板，斑块可以拿下。蓝色斑块含有一个假定的突变紫胶，我携带的噬菌体，而清晰的斑块怀有非突变体。蓝斑的频率（在日ë明确的）指示在原始细胞群的DNA从提取出的突变频率。此外，λ噬菌体托管LacI的目标可以很容易地利用PCR技术进行相对高通量测序分析。测序多个突变了LacI基因将揭示关于突变谱，这反过来又可以指向特定的DNA修复途径可能存在的缺陷，或以特定的基因毒性事件的重要信息。在LacI的转基因系统已经被标准化到多个实验室19和试剂是市售的。在LacI的系统的一个主要缺点是，检测大的缺失或重排的能力有限，因此，其他的方法， 例如 ，多色FISH对中期扩展需要被用来赞美这方面的不足。 内的LacI的小鼠模型中的λ噬菌体载体，有一个更小的基因，CII，可用于突变分析。它的大小和事实，即突变体可以选择使这比LacI的基因分析少，劳动密集和更便宜的检测20。然而，LacI的基因被更广泛地研究用于诱变21和基因突变的敏感性已被很好地表征，以便有氨基酸残基生成的显色底物22-25表型应答的清楚的理解。 其他小鼠模型进行突变检测包括使用ΦX174或LacZ基因的转基因。该ΦX174转基因小鼠模型，与原A：T→G的：C回复突变试验26或正向突变试验27，使检测碱基对替换的频谱的，代表一个成本更低的系统比LacI的模型。然而，在正向测定法的突变屏幕是不平凡的，该突变规格该ΦX174转基因的TRUM不作为良好的特点为，该LacI上的。在进行LacZ的转基因小鼠模型中，LacZ基因突变记者被回收利用E。大肠杆菌宿主细胞，以半乳糖和半乳糖含有28培养基敏感。这个系统的一个缺点是，LacZ基因靶的恢复也涉及到限制性内切酶消化后，通过连接大肠杆菌的和电穿孔大肠杆菌宿主，由此使得难以适应系统以小数量的细胞。虽然它不是用于干/祖细胞群的工作是绝对需要的（一个可以随时开始更加小鼠），如果需要大量的细胞（ 例如百万或更多）也将很快成为不切实际，成本太高。此外，LacZ基因的体积比较大，同时提供一个敏感的突变记者，繁琐，更昂贵的DNA序列分析和determ的萌发的突变谱。这种模式的主要优点但是，它能够检测到大缺失和插入，以及染色体重排。 由于所有细胞在LacI上，ΦX174和LacZ转基因小鼠模型进行的报告系统，所有这些小鼠模型可用于测量诱变在任何感兴趣的细胞类型，包括干细胞和祖细胞，只要它们能够可靠地收集以及足够数量的。因为我们与LacI的小鼠模型和LacI的突变分析的丰富经验，我们决定对本系统进一步诱变分析造血干细胞和祖人口。 造血组织是良好的特点在它的各个组成部分，包括长期再植干细胞，这是识别成霖的极为罕见人口的细胞表面表型方面- IL7R <suP&gt; -的Sca-1 + CKIT + +（LSK）/ FLK2 - CD150 + CD48 -细胞29。 。Mohrin等[4]表明，LSK/Flk2的稍大人口-细胞仍是良好的造血干细胞代表和显著的不同从最原始的承诺髓系祖（CMP）的人口，当涉及到研究DNA修复。此外，当HSC-富集LSK（的Flk-2 +和的Flk-2 - ）细胞相比，林- IL7R -的Sca-1-CKIT + +（LS-K祖细胞），但仍然是在一个显著差异NHEJ低纯度的能力5，干细胞富集的LSK人口和祖细胞。在我们的研究中，我们使用的HSC富集的LSK（的Flk-2 +和的Flk-2 - ）细胞，因为我们发现至少有2×10 5细胞都需要一致的，可靠的结果，在这个突变检测，这细胞数目是非常困难的取得时1排序LSK/Flk2 - CD150 + CD48人口甚至LSK/Flk2 -人口（在小鼠中，成本和实用性方面）。此协议的基础上，最初由Kohler 等人开发的18一详细介绍了如何自发的DNA突变频率可以在LSK细胞决定和分化的定义髓系细胞群体以及未分离的骨髓和脾脏细胞。

<table border="0" cellpadding="0" cellspacing="0" width="691">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">LacI transgenic mice&#160;</td>
			<td style="width:245px;">BioReliance Corporation</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:252px;">RecoverEase DNA Isolation Kit, including the RNace-It ribonuclease cocktail</td>
			<td style="width:245px;">Agilent Technologies (Stratagene)</td>
			<td style="width:195px;">720202</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:252px;">Transpack Packaging Extract, including the orange and blue transpack tubes and the SCS-8 E. coli</td>
			<td style="width:245px;">Agilent Technologies (Stratagene)</td>
			<td style="width:195px;">200221</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA size standards &#8211; lambda ladder</td>
			<td>Bio Rad</td>
			<td>170-3635</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.025 mM pore size membranes&#160;</td>
			<td>Fisher (Millipore)</td>
			<td>VSWP 025 00</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">245 mm2 bioassay dishes (trays)&#160;</td>
			<td>Fisher (Corning)</td>
			<td>07-200-600</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NZY broth (powder)</td>
			<td>Fisher (Teknova)</td>
			<td>N1144</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>Fisher</td>
			<td>BP1423-500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Fisher</td>
			<td>E-3120-500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N, N-dimethylformamide</td>
			<td>Fisher</td>
			<td>AC34843-5000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-gal</td>
			<td>Research Products Intl Corp (RPI)</td>
			<td>B71800-10.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase K</td>
			<td>Roche</td>
			<td>3-115-852</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR Extender Taq Polimerase kit</td>
			<td>5 PRIME</td>
			<td>2200500</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Agencourt AMPure XP cleanup kit</td>
			<td>Beckman/coulter</td>
			<td>A63880</td>
		</tr>
	</tbody>
</table>

identifying dna mutations in purified hematopoietic stem/progenitor cells

在最近几年，已经很明显，基因组不稳定性是紧密相关的许多发育障碍，癌症和衰老。由于干细胞有责任确保整个生命组织的平衡和修复，这是合理的假设，该干细胞群是维护组织的基因组的完整性至关重要。因此，显著的兴趣已经出现在评估内源性和环境因素对干细胞基因组的完整性及其后代的影响，旨在了解干细胞基础疾病的病因。 LacI的转基因小鼠携带一个可恢复的λ噬菌体载体编码了LacI报道系统，其中的了LacI基因作为突变记者。突变了LacI基因的结果是产生β-半乳糖苷酶能够水解显色底物，将它的蓝色。该LacI的报告系统是我进行n上的所有细胞，包括干细胞/祖细胞，并可以很容易地回收并用于随后感染大肠杆菌大肠杆菌 。培养大肠杆菌感染后， 大肠杆菌琼脂糖包含正确基板，斑块可以拿下;蓝斑表明突变了LacI基因，同时明确斑块怀有野生型。蓝色（其中清除）斑块的频率指示在原始细胞群的DNA从提取出的突变频率。测序突变了LacI基因将会显示突变的基因中的位置和突变的类型。 该LacI的转基因小鼠模型是行之有效的体内突变检测。此外，小鼠和试剂用于测定是市售的。在这里，我们详细描述了如何这种模式可以用来测量自发发生的DNA突变在干细胞富集的林频- IL7R -的Sca-1 + CKIT +（LSK）细胞和造血系统的其他亚群。

<html> 体内突变分析测量一个罕见的事件（突变个PFU）的许多事件中（所有个PFU）。通过用少量的细胞进行测定，这是有可能的结果是相当受假阳性和假阴性结果的影响。为解决这一问题，我们进行了连续稀释的实验与普通骨髓细胞，从三个不同的动物收获。我们测定了突变体的频率在这些动物的骨髓用1.4×10 6，7.0×10 5，3.5×10 5，1.75×10 5个细胞。结果（ 图7）显示输入?...

Watch this Scientific Journal Video about Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells at JoVE.com

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells | Immunology and Infection | Video

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

这里我们描述了对小数目使用了LacI转基因小鼠模型纯化造血干细胞的体内诱变法。该LacI的基因可以分离来确定频率，位置和类型的DNA突变的自发产生或暴露于基因毒素后。

在纯化造血干/祖细胞识别DNA突变

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

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12.9K 次观看.  UT Health Science Center at San Antonio. 这里我们描述了对小数目使用了LacI转基因小鼠模型纯化造血干细胞的体内诱变法。该LacI的基因可以分离来确定频率，位置和类型的DNA突变的自发产生或暴露于基因毒素后。 

视频: Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman's disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.

Identifying Dysregulated Genes Induced by Kaposi&#039;s Sarcoma-associated Herpesvirus KSHV

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

确定失调卡波西氏肉瘤相关疱疹病毒（KSHV）诱导的基因

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

科研

免疫与感染

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

纯净小鼠的B细胞的诱导和评估类开关重组

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages.

We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

集落形成细胞（CFC）的含量对人体的造血细胞

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

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 ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Multivirus特异性T细胞的生成，预防/治疗异基因造血干细胞移植后的病毒感染

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

建立一个<em&gt;在体外</em&gt;系统来研究细胞内的行为<em&gt;光滑念珠菌</em&gt;在人THP-1巨噬细胞

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

神经干/祖细胞在小鼠EAE的慢性全身性注射

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

可视化的活细菌细胞蛋白-DNA相互作用使用光活化的单分子跟踪

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

评估小鼠浆细胞树突状细胞在淋巴集结的开发利用造血祖细胞的过继转移

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...