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><tbody><tr><td>&lt;em&gt;LacI &lt;/em&gt;transgenic mice&amp;nbsp;</td><td>BioReliance Corporation</td><td></td><td></td></tr><tr><td>RecoverEase DNA Isolation Kit, including the RNace-It ribonuclease cocktail</td><td>Agilent Technologies (Stratagene)</td><td>720202</td><td></td></tr><tr><td>Transpack Packaging Extract, including the orange and blue transpack tubes and the SCS-8 &lt;em&gt;E. coli&lt;/em&gt;</td><td>Agilent Technologies (Stratagene)</td><td>200221</td><td></td></tr><tr><td>DNA size standards &amp;ndash; lambda ladder</td><td>Bio Rad</td><td>170-3635</td><td></td></tr><tr><td>0.025 mM Pore size membranes&amp;nbsp;</td><td>Fisher (Millipore)</td><td>VSWP 025 00</td><td></td></tr><tr><td>245 mm&lt;sup&gt;2&lt;/sup&gt; Bioassay dishes (trays)&amp;nbsp;</td><td>Fisher (Corning)</td><td>07-200-600</td><td></td></tr><tr><td>NZY broth (powder)</td><td>Fisher (Teknova)</td><td>N1144</td><td></td></tr><tr><td>Agar</td><td>Fisher</td><td>BP1423-500</td><td></td></tr><tr><td>Agarose</td><td>Fisher</td><td>E-3120-500</td><td></td></tr><tr><td>N,N-Dimethylformamide</td><td>Fisher</td><td>AC34843-5000</td><td></td></tr><tr><td>X-gal</td><td>Research Products Intl Corp (RPI)</td><td>B71800-10.0</td><td></td></tr><tr><td>Proteinase K</td><td>Roche</td><td>3-115-852</td><td></td></tr><tr><td>PCR Extender Taq Polimerase kit</td><td>5 PRIME</td><td>2200500</td><td></td></tr><tr><td>Agencourt AMPure XP cleanup kit</td><td>Beckman/Coulter</td><td>A63880</td><td></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

鉴定纯化造血干细胞/祖细胞中的 DNA 突变 |免疫学和感染|视频

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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13.0K 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

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 ...

科研

免疫与感染

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is inherently a single-cell assay, however prior to molecular analysis, the target cells are often prospectively isolated in bulk, thereby losing single-cell resolution. Single-cell gene expression analysis provides a means to understand molecular differences between individual cells in heterogeneous cell populations. In bulk cell analysis an overrepresentation of a distinct cell type results in biases and occlusions of signals from rare cells with biological importance. By utilizing FACS index sorting coupled to single-cell gene expression analysis, populations can be investigated without the loss of single-cell resolution while cells with intermediate cell surface marker expression are also captured, enabling evaluation of the relevance of continuous surface marker expression. Here, we describe an approach that combines single-cell reverse transcription quantitative PCR (RT-qPCR) and FACS index sorting to simultaneously characterize the molecular and immunophenotypic heterogeneity within cell populations.
In contrast to single-cell RNA sequencing methods, the use of qPCR with specific target amplification allows for robust measurements of low-abundance transcripts with fewer dropouts, while it is not confounded by issues related to cell-to-cell variations in read depth. Moreover, by directly index-sorting single-cells into lysis buffer this method, allows for cDNA synthesis and specific target pre-amplification to be performed in one step as well as for correlation of subsequently derived molecular signatures with cell surface marker expression. The described approach has been developed to investigate hematopoietic single-cells, but have also been used successfully on other cell types.
In conclusion, the approach described herein allows for sensitive measurement of mRNA expression for a panel of pre-selected genes with the possibility to develop protocols for subsequent prospective isolation of molecularly distinct subpopulations.

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

一种组合单细胞法表征人茎和祖种群的分子和免疫表型异质性

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

遗传学

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to reestablish blood cell production for the lifespan of the recipient. This protocol explains how to set up a functional assay to compare the HSC capacities of two different populations of cells, such as bone marrow from mice of two different genotypes, and how to analyze the recipient mice by flow cytometry. The protocol uses HSC equivalents rather than cell sorting for standardization and discusses the advantages and disadvantages of both approaches. We further discuss different variations to the basic protocol, including serial transplants, limiting dilution assays, homing assays and non-competitive transplants, including the advantages and preferred uses of these varied approaches. These assays are central for the study of HSC function and could be used not only for the investigation of fundamental HSC intrinsic aspects of biology but also for the development of preclinical assays for bone marrow transplant and HSC expansion in culture.

This protocol provides step-by-step guidelines for setting up competitive mouse bone marrow transplant experiments to study hematopoietic stem/progenitor cell function without prior purification of stem cells by cell sorting.

竞争性移植评价造血干细胞健身

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to ...

发育生物学

Characterizing Mutational Load and Clonal Composition of Human Blood

Hematopoietic stem and progenitor cells (HSPCs) gradually accumulate DNA mutations during a lifespan, which can contribute to age-associated diseases such as leukemia. Characterizing mutation accumulation can improve understanding of the etiology of age-associated diseases. Presented here is a method to catalogue somatic mutations in individual HSPCs, which is based on whole-genome sequencing (WGS) of clonal primary cell cultures. Mutations that are present in the original cell are shared by all cells in the clonal culture, whereas mutations acquired in vitro after cell sorting are present in a subset of cells. Therefore, this method allows for accurate detection of somatic mutations present in the genomes of individual HSPCs, which accumulate during life. These catalogues of somatic mutations can provide valuable insights into mutational processes active in the hematopoietic tissue and how these processes contribute to leukemogenesis. In addition, by assessing somatic mutations that are shared between multiple HSPCs of the same individual, clonal lineage relationships and population dynamics of blood populations can be determined. As this approach relies on in vitro expansion of single cells, the method is limited to hematopoietic cells with sufficient replicative potential.

Somatic mutation patterns in cells reflect previous mutagenic exposure and can reveal developmental lineage relationships. Presented here is a methodology to catalogue and analyze somatic mutations in individual hematopoietic stem and progenitor cells.

人体血液的突变载荷和克隆成分的特征

Hematopoietic stem and progenitor cells (HSPCs) gradually accumulate DNA mutations during a lifespan, which can contribute to age-associated diseases such ...

Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell disease. This observation has tremendous implications, as subsequent therapeutic immunomodulation strategies can improve clinical outcome. Conventionally, polymerase chain reaction-based analysis of short tandem repeats is used to identify chimerism in donor-derived blood cells. However, this method is restricted to nucleated cells and cannot distinguish between dissociated single-cell lineages. We applied the analysis of short tandem repeats to flow cytometric-sorted hematopoietic progenitor cells and compared this with the analysis of short tandem repeats obtained from selected burst-forming unit - erythroid colonies, both collected from the bone marrow. With this method we are able to demonstrate the different proliferation and differentiation of donor cells in the erythroid compartment. This technique is eligible to complete current monitoring of chimerism in the stem cell transplant setting and thus may be applied in future clinical studies, stem cell research and design of gene therapy trials.

Quantification of donor-derived cells is required to monitor engraftment after stem cell transplantation in patients with hemoglobinopathies. A combination of flow cytometry-based cell sorting, colony formation assay, and subsequent analysis of short tandem repeats may be used to assess the proliferation and differentiation of progenitors in the erythroid compartment.

Hemoglobinopathies 患者造血干细胞移植后残余供红祖细胞的检测

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell ...

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional properties such as proliferation and differentiation, thereby promoting the development of hematologic malignancies. Efficient and precise genetic manipulation of HSPCs is required to model, characterize, and better understand the functional consequences of recurrent somatic mutations. Mutations can have a deleterious effect on a gene and result in loss-of-function (LOF) or, in stark contrast, may enhance function or even lead to novel characteristics of a particular gene, termed gain-of-function (GOF). In contrast to LOF mutations, GOF mutations almost exclusively occur in a heterozygous fashion. Current genome-editing protocols do not allow for the selective targeting of individual alleles, hampering the ability to model heterozygous GOF mutations. Here, we provide a detailed protocol on how to engineer heterozygous GOF hotspot mutations in human HSPCs by combining CRISPR/Cas9-mediated homology-directed repair and recombinant AAV6 technology for efficient DNA donor template transfer. Importantly, this strategy makes use of a dual fluorescent reporter system to allow for the tracking and purification of successfully heterozygously edited HSPCs. This strategy can be employed to precisely investigate how GOF mutations affect HSPC function and their progression toward hematological malignancies.

Novel strategies to faithfully model somatic mutations in hematopoietic stem and progenitor cells (HSPCs) are necessary to better study hematopoietic stem cell biology and hematological malignancies. Here, a protocol to model heterozygous gain-of-function mutations in HSPCs by combining the use of CRISPR/Cas9 and dual rAAV donor transduction is described.

工程人造血干细胞和祖细胞中的致癌杂合功能获得突变

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional ...

癌症研究

Evaluation of Abnormal Growth-related Genes of Hematopoietic Stem and Progenitor Cells by Combining CRISPR/Cas9 Technology with Cell Counting

Hematopoietic stem cells possess the ability for long-term self-renewal and the potential to differentiate into various types of mature blood cells. However, the accumulation of cancerous mutations in hematopoietic stem and progenitor cells (HSPCs) can block normal differentiation, induce aberrant proliferation, and ultimately lead to leukemogenesis. To identify and/or evaluate the cancerous mutations, we integrated CRISPR/Cas9 technology with the Cell Counting Kit-8 (CCK-8) assay to investigate a model gene Trp53, that is essential for abnormal proliferative ability of HSPCs. Specifically, bone marrow cells enriched for HSPCs from Cas9 mice were harvested and then subjected to viral transfection with single-guide RNAs (sgRNAs) targeting one or several candidate genes to introduce genetic alterations in HSPCs. Then, a CCK-8 assay was performed to investigate the proliferative capacity of transfected HSPCs. The sgRNA targeting efficiency was confirmed by a Tracking of Indels by Decomposition assay. These identified genes may play crucial roles in leukemogenesis and could serve as potential therapeutic targets.

Here, we present a protocol that integrates CRISPR/Cas9 technology with the Cell Counting Kit-8 (CCK-8) assay to identify candidate genes that are crucial for the abnormal proliferative capacity of hematopoietic stem and progenitor cells.

将 CRISPR/Cas9 技术与细胞计数相结合评估造血干细胞和祖细胞生长相关基因的异常

Hematopoietic stem cells possess the ability for long-term self-renewal and the potential to differentiate into various types of mature blood cells. ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

小鼠骨髓 Oligopotent 和世系髓样祖细胞的鉴定与分离

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

生物学

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able to generate large datasets, necessitate the destruction of the cell of interest, making it impossible to assess its functional fate. Heterogeneous biological differentiating systems like hematopoiesis are therefore difficult to describe. Building on cell division tracking dyes, we further developed a protocol to simultaneously determine kinship, division number, and differentiation status for many single hematopoietic progenitors. This protocol allows the assessment of the ex vivo differentiation potential of murine and human hematopoietic progenitors, isolated from various biological sources. Moreover, as it is based on flow cytometry and a limited number of reagents, it can quickly generate a large amount of data, at the single-cell level, in a relatively inexpensive manner. We also provide the analytical pipeline for single-cell analysis, combined with a robust statistical framework. As this protocol allows the linking of cell division and differentiation at the single-cell level, it can be used to quantitatively assess symmetric and asymmetric fate commitment, the balance between self-renewal and differentiation, and the number of divisions for a given commitment fate. Altogether, this protocol can be used in experimental designs aiming to unravel the biological differences between hematopoietic progenitors, from a single-cell perspective.

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Presented here is a flow cytometry-based technique that allows for simultaneously measuring the number of cell divisions, surface cell phenotype, and cellular kinship. Those properties can be tested statistically using a permutation-based framework.

通过流式细胞术 同时 评估造血干细胞和祖细胞的亲缘关系、分裂数和表型

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able ...

在纯化造血干/祖细胞识别DNA突变

摘要

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此视频中的章节

集落形成细胞（CFC）的含量对人体的造血细胞

一种组合单细胞法表征人茎和祖种群的分子和免疫表型异质性

竞争性移植评价造血干细胞健身

人体血液的突变载荷和克隆成分的特征

Hemoglobinopathies 患者造血干细胞移植后残余供红祖细胞的检测

工程人造血干细胞和祖细胞中的致癌杂合功能获得突变

将 CRISPR/Cas9 技术与细胞计数相结合评估造血干细胞和祖细胞生长相关基因的异常

小鼠骨髓 Oligopotent 和世系髓样祖细胞的鉴定与分离

表型分析和分离小鼠造血干细胞和传承的承诺祖

通过流式细胞术同时评估造血干细胞和祖细胞的亲缘关系、分裂数和表型

在纯化造血干/祖细胞识别DNA突变

摘要

探索更多视频

此视频中的章节

集落形成细胞（CFC）的含量对人体的造血细胞

一种组合单细胞法表征人茎和祖种群的分子和免疫表型异质性

竞争性移植评价造血干细胞健身

人体血液的突变载荷和克隆成分的特征

Hemoglobinopathies 患者造血干细胞移植后残余供红祖细胞的检测

工程人造血干细胞和祖细胞中的致癌杂合功能获得突变

将 CRISPR/Cas9 技术与细胞计数相结合评估造血干细胞和祖细胞生长相关基因的异常

小鼠骨髓 Oligopotent 和世系髓样祖细胞的鉴定与分离

表型分析和分离小鼠造血干细胞和传承的承诺祖

通过流式细胞术 同时 评估造血干细胞和祖细胞的亲缘关系、分裂数和表型

通过流式细胞术同时评估造血干细胞和祖细胞的亲缘关系、分裂数和表型