This research was funded by the Canadian Regulatory System for Biotechnology (CRSB) and Chemicals Management Plan (CMP) initiatives.

所有涉及畜牧业，维护和处理方案批准了加拿大卫生部的动物保健委员会。 1，动物暴露<ol><li>随机分配转基因雄性小鼠（8-12周龄）通过选定用药时间适当的暴露途径，控制组和治疗组（分= 5元组）.Treat小鼠试验化合物和相应的控制。根据所关注的精细胞类型（ 图3）选择适当的采样时间。 </li><li>后的采样时间，由下异氟烷麻醉颈脱位（或其它适当的方法）安乐死的小鼠。小心地从腹部或阴囊切口吸取睾丸切除马尾附睾。（ 图4，查看附睾收集了详细的视频中看到Duselis 等 24 条）。另外，收集睾丸，如果分析生精小管细胞。冷冻在液氮中，并储存于-80℃以备后用。 </li></ol> 2，隔离消化马尾精子和<ol><li>解冻冰副睪尾。转让解冻马尾到培养皿中，用手术刀或刀片彻底剁碎。 </li><li>加入700μl室温下的D-PBS中的培养皿。通过绘制和发布停牌，直至D-PBS变得混浊精子（约10倍），使用大口径千微升枪头，释放精子的马尾。注意：为了制备大孔移液管尖端切成2-3毫米的由塑料移液管尖的末端。 </li><li>通过不锈钢网过滤器过​​滤悬浮到新的1.5 ml管。一个额外的700微升的D - PBS冲洗培养皿，并通过网状过滤器转移到同为1.5 ml管。除去网状，将管置于冰上。 </li><li>重复步骤2.1-2.3剩余的样品。 </li><li>旋samplesat 11000 XG 3分钟。小心倒出上清液。避免打扰沉淀。 </li><li>加入1.0毫升冷的1×柠檬酸盐钠（SSC）。漩涡，直到沉淀完全重悬。这有时需要数轮涡旋。 </li><li>加入15微升10％的SDS。反相/剧烈振荡30秒，以破坏非精子细胞。振摇过轻轻将导致破坏不足，并在以下步骤中的小球将无法正常形成。 </li><li>旋在11000×g离心2分钟。一松，“蓬松的”颗粒表明体细胞的不完全中断，由于2.7步摇不足的。如果发生这种情况，只需再次撼动样品，并重新流片之前紧张的沉淀形成的。小心地倒出上清液。避免打扰沉淀。简言之再次旋转，并与200微升移液器取出剩余的上清液。 </li><li>加入940微升0.2倍冷SSC和涡直到沉淀重悬。该颗粒可以是非常困难的重新悬浮并可能需要多轮涡旋。第有时团块烫发是不可避免的。 </li><li>加入120微升β-巯基乙醇，加入100μl10％SDS中，20微升的0.5M EDTA，pH为8，和20μl蛋白酶K（60毫克/毫升，新鲜制备）。搅拌均匀，用旋转消化过夜37℃。继续进行酚/氯仿抽提。 </li></ol> DNA的马尾精子三酚/氯仿抽提注意：由于在晚期精子细胞和成熟精子核DNA络合与鱼精蛋白，并且是高度浓缩的相比，体细胞的DNA中，常规的核酸分离方法将不会产生足够的产率和纯度的突变检测，以变通的DNA有效。需要积极消化后多酚-氯仿抽提释放 ​​，净化精子DNA（根据方法15）。 <ol><li>转让精子细胞消化到15毫升聚丙烯管。 </li><li>加入2mL苯酚：氯仿混合物（1:1）。旋转管22转3分钟。 </li><li>离心机在1600×g离心10分钟，并且随着模糊界面层转移水性顶层到新鲜的15ml试管中。 </li><li>重复步骤3.1.2和3.1.3的3倍，但改变旋转倍至3分钟，4分钟，6分钟，分别。在最后重复，避免传输任何“模糊”的界面层。 </li><li> 第 4 次萃取后，加入70微升3M醋酸钠，pH值5.2每1ml水提取物和2毫升的氯仿中：异戊醇（24：1）。旋转管在22转12分钟。 </li><li>离心机在1600×g离心10分钟，含水顶层转移到新的15ml试管中。 </li><li>通过加入2倍体积的无水乙醇中，然后轻轻旋转在其一侧轻轻摇动试管沉淀DNA。 </li><li>通过后台到热封牧场吸管尖收集的DNA。纷飞70％的乙醇和空气干燥的枪头5分钟冲洗的DNA。 </li><li>萃取后，溶解在40的DNA沉淀 - 100微升三S-EDTA缓冲液，pH为8，贮存在4℃。允许该DNA，以在4℃下进到lacZ基因突变试验前溶解了至少两天。如果溶解度问题都遇到过，DNA可以进一步溶解在65℃下在使用前15分钟。确定与spectrophotometre中的DNA的浓度在A 260，并确保溶解DNA的浓度为200-2,000纳克/微升之间。 </li></ol> 4，隔离从生精小管生殖细胞和消化<ol><li>如果冻结，解冻睾丸冰（约1小时）。睾丸转移到毛玻璃板。 </li><li>保持睾丸的一端有一对镊子。在睾丸的另一端，穿刺用另一对镊子或一对解剖剪刀（ 图5A）中的上皮细胞胶囊的孔。通过穿刺挤压生精小管并弃去上皮胶囊（ 图5B）。 </li><li> áDD 500微升室温D-PBS的解封装后的曲细精管。 </li><li>角的组织辊（硅酮橡胶紧紧地装配在自由转动的直径为5mm的不锈钢管，或类似装置），使得一端是在5-10°（ 图5C）的近似角度与板接触。不施加任何压力，轻轻移动辊来回的小管，直到它们被压平，并在D-PBS变得浑浊和释放的细胞（约5到10倍）。 </li><li>添加另一个500微升的D-PBS过的小管，轻轻翻转肾小管一些额外的时间。 </li><li>细胞悬液转移到1.5 ml离心管，同时尽量减少脱落的肾小管被转移量（ 图5D）。 </li><li>重复步骤4.5-4.6，如果需要收集更多的细胞。 </li><li>让1 - 2分钟为任何偶然收集细管沉降到管底。传输的D-PBS为FRESH1.5毫升管离开落户小管（大约100微升的D - PBS）的后面。一小等分的该悬浮液可以用显微镜（相位相反）下进行检查，以评估细胞群的组合物。 </li><li>降速细胞在11000 XG 30秒。小心地倒出上清液，而不会干扰沉淀。将细胞沉淀物如果需要，可以冷冻在-80℃，在这一点上。 </li><li>如果需要解冻的细胞。转移至15ml聚丙烯管和悬浮细胞在5ml裂解缓冲液（10mM的Tris pH值7.6，10mM的EDTA，100mM的氯化钠，1毫克/毫升的蛋白酶K，1％SDS）。摘要过夜孵化器与旋转在37℃。 </li><li>继续进行酚/氯仿抽提。 </li></ol> 5，苯酚的DNA /氯仿提取曲细精管生殖细胞注意：A不太积极的提取是用于分离生精小管精原细胞的DNA中，由于这些类型的细胞还没有到n进展uclear凝结。 <ol><li>加入5 mL酚：氯仿混合物（1:1）过夜曲细精管细胞消化。旋转筒22旋转20分钟。 </li><li>离心机在1600×g离心10分钟，并传送含水顶层，同时避免了“模糊”的界面层，以新鲜的1​​5ml试管中。 </li><li>加入100μl5 M氯化钠每5ml水提取物（通常是5毫升恢复） </li><li>加入5 ml的氯仿：异戊醇（24：1）。旋转管在22转12分钟。 </li><li>离心机在1600×g离心10分钟，含水顶层转移到新的15ml试管中。 </li><li>通过加入2倍体积的无水乙醇中，然后轻轻旋转和反转试管沉淀DNA。 </li><li>通过后台到封闭牧场吸管尖收集的DNA。纷飞70％的乙醇和空气干燥的枪头5分钟冲洗的DNA。 </li><li>在40-100微升的Tris-EDTA缓冲液，pH为8，贮存在4℃下溶解的DNA。允许的DNA在4℃溶解了至少两个在进行lacZ的突变试验前几天（见步骤3.9）。如果溶解度问题都遇到过，DNA可以进一步溶解在65℃下在使用前15分钟。确定与spectrophotometre中的DNA的浓度在A 260，并确保该浓度是200至2000毫微克/微升。 </li></ol> 6 LacZ基因突变试验细菌或真菌污染可以与包装效率，以及斑块生长和得分干扰。它使用适当的无菌措施，以防止包装反应，宿主培养和生长培养基的污染进行lacZ基因测定法，因此至关重要的。 <ol><li>天测定前：准备底部琼脂和过夜培养<ol><li>八板（4板得分突变，4板来算滴度）装有8毫升底部琼脂每个都是每个样品需要（ 即 ，64毫升每个样品）。底部琼脂是相同的两个该突变体和效价数板。准备足够底部琼脂用于被处理的样本的数目。在无菌条件下倾为90毫米的培养皿中（每个培养皿8ml）和允许琼脂固化。注：底部琼脂平板上，可以制备多达提前1周。 </li><li>在50ml管中加入10 ml的LB肉汤中，将100μl的20％麦芽糖溶液中，加入25μl氨苄青霉素（20毫克/毫升）和20μl的卡那霉素（5毫克/毫升）。接种了大肠杆菌大肠杆菌 （lacZ的- /盖尔- ）25振荡以240rpm一夜长大，在37℃。 </li></ol></li><li>第1天：亚文化细胞<ol><li>子培养细胞制备1:100稀释在新鲜LB（无抗生素）的过夜培养物。为8ml继代培养的体积，需要每个样品。在37℃下用在240 rpm振摇约3.5小时，直到OD 600 = 1。 </li><li>当OD 600 = 1，分细胞悬浮液均匀地入50ml试管并离心1300 XG在15℃下进行10分钟。除去上清液，并在半（ 即 ，每样品4毫升）含10mM 硫酸镁的LB原体积的重新悬浮细胞。把电池放在一边，直到需要使用步骤6.2.4.3。 </li></ol></li><li>第1天：包装DNA的λ噬菌体颗粒<ol><li>暖水浴30℃。 </li><li>采用大口径10微升枪头，转移4微升的DNA，以1.5 ml管。注意：此步骤可以进行一天或更提前，以减少制备时间。 </li><li>热身从噬菌体包装提取试剂盒的第一管（红色）（1筒，每2个样本）。短暂离心管在底部收集提取物。 </li><li>转让4.8微升包装提取物从第一管的DNA样品，并通过温和搅拌和枪头混匀。简要降速样品管。孵育1.5小时，在30℃水浴中。 </li><li>热身第二个（蓝色）包装提取管（1管〜15个样品）。短暂离心收集提取物底部管。 </li><li>转让4.8微升包装提取物从第二管的DNA样品，并轻微搅拌混合。简要降速样品管。孵育额外的1.5小时，在30℃水浴中。 </li><li>重悬在500μl的SM缓冲包装的噬菌体颗粒并通过在20rpm下旋转30分钟混合。 </li><li>后旋转时，简要地涡旋样品并离心在11000×g离心30秒收集样品在管的底部。噬菌体颗粒是准备感染[步骤6.2.4〕。 </li></ol></li><li>第1天：准备顶层琼脂<ol><li>准备单独的顶层琼脂的滴度板和突变体筛选板。每个样本需要4滴定板，和4突变体板。每块板需要8毫升顶层琼脂。加入选择性试剂苯基-β-D-吡喃半乳糖苷（p-半乳糖）的突变体的选择顶层琼脂只。准备两个顶部琼脂提前（测定法的第二天），并在50℃下加入硫酸镁 ，以两个顶部琼脂之前保持和PG人到突变体筛选琼脂。 </li></ol></li><li>第1天：感染细胞与包装噬菌体和电镀<ol><li>标签两50ml试管，每个样品：1“突变”管每个样品，每个样品1“效价”管</li><li>标签8琼脂每个样品板：每个样品4“突变体”板块，每个样品4“效价”板块</li><li>等分2毫升悬浮细胞[从步骤6.2.1.2]对各管。 </li><li>加入500μl包装的噬菌体颗粒[步骤6.2.2.8]为“突变”50ml管中（含电池）的。轻轻混合，并允许噬菌体颗粒感染细胞30分钟，在室温下进行。 </li><li> 30分钟后，短暂涡​​旋感染的细胞和15微升感染细胞转移到适当50毫升“滴度”管（包含细胞）。 </li><li>板块滴度样品，加入30毫升温水（50℃）“效价”顶琼脂（不含P-半乳糖），以“效价”50ml管中。立即DISTRIB4“滴度”板之间尤特琼脂/细胞混合物（〜每板8毫升）中。工作很快使最高琼脂没有清凉的移液管，并尽量不要引入气泡。 </li><li>盘子旁边的“突变”的样本。确保P-半乳糖被添加到“突变选择”顶琼脂。加入30毫升的温“突变体筛选”琼脂（含P-半乳糖），以“突变”50ml管中。立即分发4“突变体”板中的琼脂/细胞混合物（〜每板8毫升）中。 </li><li>允许板以固化（约15分钟），然后翻转并在37℃下过夜。 </li></ol></li><li>第2天：计算斑块<ol><li>过夜培养后，计数在突变体和滴定板的噬菌斑数目。对于大量斑块，计数板的仅一部分来估计的总计数（ 例如 ，对滴定板的常¼将具有100-200之间的斑块）。最低100斑块应每板时，腠算做nting的板的一部分。 </li><li>计算的噬斑形成单位（PFU就能）每微升的细胞数。这是通过将噬斑上的“效价”板的数目由细胞接种体积（15微升）进行。 </li><li>使用个PFU数/μl的估计在感染的细胞铺在该“突变体”板（个PFU /微升*总容积个PFU的总数[2毫升细胞+0.5毫升包装的噬菌体颗粒 - 15微升为滴定板] ）。 </li><li>通过将在从“效价”平板测定感染细胞的总体积计的4“突变体”板由个PFU的总数估计突变斑的总数估计MF。 </li></ol></li><li>当自发的lacZ MF是在3×10 -5对照组，秩序，因为它的MutaMouse生殖细胞，经合组织指导方针建议最少每头动物是屏幕125,000 300,000非突变个PFU为了得到可靠的基准信号编为突变。其他转基因模型可以具有较低的自发MF，在这种情况下，将需要更大的数个PFU的。一个统计功率测试可以确定个PFU和动物，以获得所需的分辨率所需的最小数目来执行。从多个复制的数据可以被合并，以满足该最小PFU的要求，条件是它们不产生显著不同突变体的频率。 </li></ol> 7统计实验装置的分析是鼠标。从该试验中产生的数据通常不是正态分布。因此，选择基于数据的分布特性进行分析的统计方法。 注意：如果相应的数据转换应用于均衡响应的方差横跨observa的范围标准参数的分析（ 例如 ，ANOVA），也可以采用化。泊松或二项式回归分析往往是比较合适的。非参数的分析也可以使用。我们经常使用广义线性模型过程采用泊松回归（ 即 ，PROC GENMOD）的SAS v.9.2（SAS软件研究所，凯里，北卡罗来纳州）所描述的勒米厄等[26]。

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The authors declare that they have no competing financial interests.

与传统方法相比，三峡水库生殖细胞突变试验提供了一种更快，更经济和更灵敏的定量诱导体内的生殖细胞基因突变的方法。通过直接在精子评估转基因MF，相对于后代的动物的数量，时间和评估的任何单个化合物的种系突变性所需的资源是显著降低。在感光度方面，我们能够探测到下面每使用剂量组只有5只动物暴露在25毫克/公斤ENUà显著2.6倍的精原干细胞的MF。相反，在特定的基因座检测无法检测到使用中的MF任何显著变化在此相同剂量&gt; 3000小鼠暴露组和&gt; 500,000对照小鼠28。 除了它的适用性都机械和监管调查，此方法提供了一种体细胞和生殖系穆塔蒂之间进行比较研究的机会对利率。最近的证据表明，对某些药物的生殖细胞突变可以在较低的浓度来诱导比所需的体细胞突变。例如，长期暴露到N-羟甲基丙烯酰胺，食品致癌物丙烯酰胺12的代谢物，增加的显性致死性生殖细胞突变在小鼠中的频率而不影响微核频率中的红血细胞，一个传统的计量体细胞遗传损伤13。此外，小鼠无论是主流烟和侧流烟雾上升的原因突变频率在精子串联重复序列的DNA位点的暴露剂量不增加血液微核率14。这些发现挑战了体细胞基因毒性测试始终是保护生殖细胞的假设，并加强对量化生殖细胞基因突变频率的更有效率和成本效益的方法的需求。然而，对于优惠的生殖细胞诱变的证据仍然薄弱，主要是由于缺乏可用的数据，在体细胞和生殖细胞的组织比较突变率。在TGR突变试验允许并行测试和诱导突变率在使用相同转基因的多个组织进行比较。因此，使用三峡水库试验将有助于填补周边的优惠生殖细胞影响的可能性的数据差距比较突变检测。 体细胞和生殖细胞突变监管测试评估同时也将通过减少所需动物的数量提高了工作效率。经合组织的准则，体细胞突变建议28天的管理时间，随后进行了三天的采样时间（28 +3）。马尾精液分析可以提供在这个时间点的敏感性较差，因为它的目标细胞精子发生过程中的精母细胞和精子细胞阶段（ 图3）大多暴露出来。在这些阶段的细胞不合成的DNA，并逐渐失去其对DNA修复6容量&lt;/ SUP&gt;。此外，采样马尾精子在这个时间点将会失败检测的精原细胞和干细胞中发生的突变。因此，融入28 +3的设计，经合组织指导方针建议的曲细精管收集的生殖细胞。此混合种群含有DNA合成和修复熟练的细胞类型，包括干细胞来源的细胞，并且在绝大多数精相被暴露。然而，由于这些细胞的混合性质，生精小管细胞分析不提供相位特定的信息。此外，有人担心非靶细胞的存在可以影响所观察到的中频（ 例如假阳性生殖细胞突变的呼叫由于从DNA修复缺陷型生殖细胞污染的突变体细胞，或稀释的突变生殖细胞信号） 。目前没有足够的数据来断定是否从曲细精管细胞成绩在28±3提供相同的敏感性和特异性Š马尾精子在稍后的时间点。我们的实验室目前正在比较各采样时间后，收集到的解决了这一点曲细精管细胞和马尾精子诱导的MF。我们注意到，OECD指导方针建议的28天为慢慢除以组织替代采样时间如肝脏也可以适合于生殖细胞的分析。然而，现有的数据仍不够，我们目前无法建议一个单一的实验设计的体细胞，并利用监管测试的TGR突变试验生殖细胞的同时分析。 该测定中应该注意的一个特征是，突变事件是在非转基因小鼠进行评估。但是，有充分的证据表明，转基因响应环境诱变剂以类似的方式来内源基因20。此外，由于独立的突变事件的确切来源难以解析，结果通常报告为突变频率（相对于突变频率）。实际的突变频率可如果结果被用于校正克隆扩增的DNA测序（即可以向转基因的突变体所观察到的频率的单个突变的细胞，即 ，除法和乘法）来解决。该突变体转基因的测序也可以进行以表征的lacZ基因突变，并确定可能从克隆扩增事件衍生的突变体，尽管这显著增加了时间和分析的成本。除了​​lacZ基因，theλgt10转基因载体包含一个替代温度依赖性突变的报告基因：在λCII基因，它是较短的（294 bp的比的3021 bp的lacZ基因 ， 图1），更容易进行排序29的一种变型。测序也允许诱发突变谱的分析，提供了洞察mutatio所讨论的化合物的最终机制。克隆扩增的一个极端的例子是一个“大奖”突变的发生（ 即 ，基因突变器官发展的一个非常早期的阶段，有助于大幅提升中频，有时数百次大于背景：千美元）。动物或组织以“中奖”的突变应该从分析中删除。 我们已经描述的测定是广泛地适用于其他TGR模型，诸如BIGBLUE小鼠和大鼠，和lacZ启动质粒鼠标，所有这些怀有类似突变报告基因载体（在20中综述）。生殖细胞的研究绝大多数迄今为止所进行的雇用类似的方法也几乎全部集中在很好地表征诱变剂如ENU和辐射（30审阅）。据预计，与最近发布的OECD测试指引，三峡水库试验中，此法会日益流行的化学筛选和监管评价。结合三峡水库生殖细胞突变试验向调节测试电池将被允许诱变的有效评估，生殖细胞11填补现有的差距。另外，该测定可用于测量MF在几乎任何组织，提供一个合适的装置，用于由环境因素在相同遗传终点的体细胞和生殖细胞的相对灵敏度进行比较，以突变的诱导。

在生殖细胞零星的DNA突变可导致生殖能力降低，如果继承的，可能会导致遗传性疾病或加剧倾向癌症的后代1-3。大量证据表明， 从头突变相当大的比重，从父系4种系遗传，而突变的后代的数目是积极与父亲年龄在怀孕5的时间相关。雄性突变比例越高被认为是两性在配子在年龄的差异的结果，更大数目与oogenic细胞分裂中的女性生殖系2的数量比生精细胞分裂，并在DNA的逐渐下降修复随着年龄的增长，男性的效率。所有的这些因素导致了复制错误的，在男性生殖系6的概率增加。然而，父系曝光的影响，以ENVIROnmental因素对新发突变的频率仍然不明朗。然而，大量的环境剂是已知的诱发生殖细胞突变，在啮齿类动物中如图7所示 ，并且有越来越多的证据表明，一些这些药剂也可能会影响人类种系8。尽管这些问题，化学物质被定期地测试他们的诱导体细胞用于管理目的的突变的能力以及它通常假设体测试是足以保护种系。因此，化学品很少评估其诱发生殖细胞突变的能力。 其中一个原因生殖细胞致突变性试验已基本从监管决策过程中忽略的是缺乏实际的方法。传统的啮齿动物为基础的方法，例如显性致死9和特定基因座10的测试中，通过计分突变体的表型的胚胎或后代估计生殖细胞突变率露的父母。这些测定需要使用非常大量的动物，时间和资源，以获得统计学上有意义的结果。 虽然一些现代的方法来量化生殖细胞突变，最近出现了很多苦的缺点在他们的实用性，高效性和生物相关的条款。例如，重复长度突变扩展简单的串联重复序列（ESTR）位点可以量化使用单分子PCR检测方法15雄性生殖细胞。但是，执行此方法可以在技术上具有挑战性的和艰苦的，而不像点突变，变化极不稳定ESTR位点的重复长度的生物和健康的重要性仍不清楚16。当应用于遗传突变4,17问题现代全基因组测序技术能提供丰富的生物学意义的数据，但成本高，高错误率，需要相关的验证确认突变和生物信息学的挑战仍然限制该选项的常规应用的监管检测能力18。 在此，我们介绍直接在转基因雄性小鼠的生殖细胞定量诱发突变的实用方法。这个协议描述了在转基因MutaMouse模型，它具有含大肠杆菌lacZ报告基因整合到染色体3 19（ 图1）的两个拷贝的重组λgt10噬菌体载体的多个链接的副本。 该协议还涉及其他的转基因啮齿动物（TGR）基于相同的原理（BIGBLUE小鼠和大鼠或lacZ基因的质粒小鼠等 ）或稍有不同的报告基因（GPT三角洲小鼠和大鼠模型中，TGR模型Lambert 等点评人，20）。这种方法是基于最近发布的说明的TGR突变试验订及经修订的OECD测试指引21，我们在适应，因为精子的独特特点突变评估在男性生殖所需要的特殊注意事项详细说明。简单地说，该法涉及揭露转基因雄性小鼠诱变物质，随后在那里预病变突变固定成稳定的突变采样时间。在选择采样时间，小鼠安乐死和生殖细胞无论从马尾附睾或输精管收集。如下面讨论的，在精子发生过程的不同阶段诱变效应可以通过选择曝光和样品采集之间的时间来确定。转基因插入物，其特征在于每个单元的λ噬菌体基因组的多个拷贝，是从生殖细胞的基因组DNA中分离并包装成空λ噬菌体衣壳产生感染性λ噬菌体颗粒随后用于感染的大肠杆菌大肠杆菌宿主。被感染的细菌生长在SELEC略去媒体认为可以区分包含与lacZ基因从细胞窝藏野生型lacZ的突变拷贝的载体细胞。暴露于男性生殖细胞的致突变作用，通过比较对照和处理的小鼠之间的突变体的转基因（ 图2，在Lambert 等。20点评）的频率来确定。大量的生殖细胞可从单个小鼠进行测定，得到比传统方法该试验中具有较高的敏感度，同时减少了所需的动物数量。而且因为没有专门的设备或训练是必需的，此法规定了生殖细胞突变检测中最现代化的毒理学/分子生物学实验室的实用和有效的选择。 针对三峡库区生殖细胞突变试验的有效应用的一个基本要求是生精周期（ 图3）的理解。时间鼠标生殖细胞从圣进展EM细胞在曲细精管的精原细胞到，精母细胞，精子细胞，最后到成熟精子在附睾内（ 即精子）是约49天。突变可发生在这个周期的各个阶段，是经常化合物特异性的。这是特别有关的诱变在雄性生殖细胞中的两个关键特性是DNA合成过程中的早期减数分裂停止，和DNA修复能力6时迟后减数分裂的渐进性丧失，所需要的诱导和固定两种工艺大多数突变。 因为精子的这些独特的特性，有三个关键实验变量的TGR生殖细胞突变试验的进行：（1）试验化合物给药时间; （2）采样时间;和（3）的细菌细胞群的选择，以收集用于分析（ 图3和表1）。管理时间的experimeNTAL变量用于确定多久靶细胞暴露于受试化合物。给药时间长度也可以用于靶向暴露于特定的细胞类型或精子发生阶段。例如，一个单一的一天给药可以用来确定对一个特定的信元类型的急性暴露的影响。类似地，接触可通过靶向仅减数分裂分割精母细胞，或有丝分裂分裂的精原细胞用2周的给药时间和适当的采样时间被聚焦到整个精子发生阶段，例如。慢性和亚慢性给药时间被用于评估长期接触的效果，以确保在测试化合物的足够的药代动力学分布，或允许从弱诱变剂突变足够的积累（例如，建议在OECD测试在28天给药时间指引）。 取样时间是用于确定关键的变量在这精子发生阶段的靶细胞中，在暴露的时间。采样时间决定了多少时间，并且因此关于进一步沿精周期，细胞通过曝光后。例如，要研究干细胞精原细胞的影响，采样时间&gt;49天要求，如果收集完全成熟的精子，或&gt; 42天内由曲细精管收集未成熟的生殖细胞，以确保所有收集到的细胞有足够的时间从暴露的开发干细胞。要注意的是至少70天的取样时间最好将表现出真正的干细胞的作用，以提供足够的时间使毒物的药代动力学分布，为消除细胞后精子发生阶段暴露的，并且考虑到这一点很重要一段时间暂时不育暴露于高致突变化合物22后，可能会出现〜6周了。同样的，21天的采样时间将确保精子科尔从马尾附睾反恐执行局也刚完成减数分裂暴露的最后一天。 生殖细胞可以收集成熟的精子由附睾马尾，或从曲细精管生精的各种细胞类型的混合物。成熟精子留在马尾为〜3天，从而能够确定与相对精度从中精子起源对于任何给定的实验设计的细胞类型精子或相位。因此，马尾精子证分析，具有高度针对性的阶段特异性突变的影响进行调查。另一方面，从生精小管收集细胞悬液包含在不同的发展阶段不同的生殖细胞类型的混合物，从而提供在其中的突变源自生精相较差的分辨率。此外，从生精小管中回收的细胞悬浮液往往含有过量的表示精子细胞中，接着精母细胞，和版Ÿ几个精原细胞和干细胞（这些比例由毕业白条如图3所示）。此外，从生精小管制备悬浮液也可以含有各种体细胞。因此，由于如此多的细胞类型都存在，突变的影响可以通过多种非靶细胞的影响。然而，从曲细精管采集样品提供了一种经济的选择同时筛选多个生殖细胞类型，并易于集成的生精细胞分析纳入经合组织的标准测试协议的体细胞突变。 重申，根据不同的研究者，给药时间，采样时间和所收集的细胞群体的需要可以调整，以询问暴露在各种细胞类型和在精子发生过程的不同阶段的影响。通过仔细地选择这些变量，实验可以被设计为靶向的机制研究，或为更广义调节测试荷兰国际集团的目的。 为了达到熟练掌握的实验中，我们建议使用100毫克/公斤N-乙基-N-亚硝基脲（ENU）急性口服，随后70天采样时间为阳性对照。马尾精子的分析从而针对精原干细胞（ 图3），其通常表现出突变频率（MF）以下ENU的这个高度诱变剂量4-5倍的增加超过对照组。但是应当注意的是，该剂量被称为诱导不育6周后曝光，因此可能不适用于较短采样时间的适当的控制剂量。这个剂量也将产生在大多数体细胞组织20可检测增加了MF。下面提出的有代表性的结果产生以下用三个剂量ENU的直至并包括100毫克/千克急性70接触途径。

<table border="0" cellpadding="0" cellspacing="0" width="1326">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Name of Reagent/Material</td>
			<td style="width:145px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td style="width:664px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MutaMouse</td>
			<td>Covance</td>
			<td>-</td>
			<td style="width:664px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">E.coli (lacZ-/galE-)</td>
			<td>Covance</td>
			<td>-</td>
			<td style="width:664px;">See reference 25 in manuscript</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform</td>
			<td>Caledon</td>
			<td>3001-2-40</td>
			<td style="width:664px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s phosphate-buffered saline (D-PBS)</td>
			<td>Gibco</td>
			<td>14190-250</td>
			<td style="width:664px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylenediaminetetraacetic acid (EDTA), 0.5M, pH 8</td>
			<td>Sigma-Aldrich</td>
			<td>03690 FLUKA</td>
			<td style="width:664px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoamyl alcohol</td>
			<td>Caledon</td>
			<td>2/10/7900</td>
			<td style="width:664px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lennon LB broth base</td>
			<td>Invitrogen</td>
			<td>12780-029</td>
			<td style="width:664px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless steel mesh filter</td>
			<td>Sigma-Aldrich</td>
			<td>S3770</td>
			<td style="width:664px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transpack packaging extract</td>
			<td>Agilent Technologies</td>
			<td>200220</td>
			<td style="width:664px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td style="width:664px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethyl alcohol, anhydrous (EtOH)</td>
			<td>Commercial Alcohols</td>
			<td>-</td>
			<td style="width:664px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Ampicilin</td>
			<td>Gibco</td>
			<td>11593-027</td>
			<td style="width:664px;">prepare 20 mg/ml in dH2O</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Kanamycin</td>
			<td>Gibco</td>
			<td>11815-024</td>
			<td style="width:664px;">prepare 5 mg/ml in dH2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenol</td>
			<td>Invitrogen</td>
			<td>15509-097</td>
			<td style="width:664px;">Saturate in 0.1 M Tris-HCl as per manufacturers direction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenyl-&#946;-D-galactopyranoside (P-Gal)</td>
			<td>Sigma-Aldrich</td>
			<td>P6501</td>
			<td style="width:664px;">dissolve 3 g per 10 ml of dimethylformamide</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Proteinase K</td>
			<td>Invitrogen</td>
			<td>25530-031</td>
			<td style="width:664px;">prepare 60 mg/ml solution dH2O just before use, 20 &#181;l per sample</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium acetate (NaAc)</td>
			<td>Fisher Scientific</td>
			<td>BP333-500</td>
			<td style="width:664px;">prepare 3M solutution, pH 5.2</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>L4390</td>
			<td style="width:664px;">prepare 10% solution in dH2O</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">1 M MgSO4</td>
			<td></td>
			<td></td>
			<td style="width:664px;">24.6 g MgSO4&#183;7H2O per 100 ml dH2O, autoclave, store at room temperature up to 1 year</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">20% maltose solution</td>
			<td></td>
			<td></td>
			<td style="width:664px;">20.0 g maltose, 24.6 g MgSO4&#183;7H2O, per 100 ml dH2O, filter sterilize, sore at 4&#7506;C up to 6 months</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;">Bottom agar</td>
			<td></td>
			<td></td>
			<td style="width:664px;">Prepare 64 ml per sample (8 pitri plates per sample X&#160; 8 ml per plate): 5.0 g LB base, 6.4 g NaCl, 7.5 g agar, per 1L dH2O. Autoclave and cool to 50&#7506;C before pouring into Petri plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LB Broth</td>
			<td></td>
			<td></td>
			<td style="width:664px;">5.0 g LB base, 6.4 g NaCl per 1L dH2O. Autoclave and cool</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline sodium citrate (SSC)</td>
			<td></td>
			<td></td>
			<td style="width:664px;">150 mM NaCl, 15 mM trisodium citrate, pH 7.0&#160;</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;">SM Buffer</td>
			<td></td>
			<td></td>
			<td style="width:664px;">5.8 g NaCl, 2.0 g MgSO4&#183;7H2O, 50 ml 1 M Tris-HCl (pH 7.5), 5.0 ml of gelatin (2% w/v), per 1L dH2O, autoclave, store at room temperature up to 1 year</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TE buffer</td>
			<td></td>
			<td></td>
			<td style="width:664px;">10 mM Tris, 1 mM EDTA, pH 8</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;">Top agar, mutant plates</td>
			<td></td>
			<td></td>
			<td style="width:664px;">Prepare 32 ml per sample (4 mutant plates per sample X 8 ml per plate): 5.0 g LB base, 6.4 g NaCl, 7.5 g agar, per 1L dH2O. Autoclave and cool to 50&#7506;C. Add MgSO4 to 10 mM</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;">Top agar, titre plates</td>
			<td></td>
			<td></td>
			<td style="width:664px;">Prepare 32 ml per sample (4 mutant plates per sample X 8 ml per plate): 5.0 g LB base, 6.4 g NaCl, 7.5 g agar, per 1L H2O. Autoclave and cool to 50&#7506;C. Add MgSO4 to 10 mM and P-Gal to 3 g/L</td>
		</tr>
	</tbody>
</table>

transgenic rodent assay for quantifying male germ cell mutant frequency

De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro positive selection assay to measure in vivo mutations induced in a transgenic &#955;gt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.

随着每只动物200000斑块的平均斑块数，我们通常用的1.7×10 -5在我们的对照组，标准差约为2.8×10 -5的雄性生殖细胞观察到的平均背景MF（基于数据来自八个独立实验）。与此菌斑计数，背景水平和方差，剂量组中n = 5只动物的每个都足以检测出2倍的MF与功率&gt; 0.8。 结果通常报告表格或图形格式。 表2和图6示出了从MutaMouse男性马尾精子组（n =每组5只）暴露于一个单一的急性口服剂量为0，25，50代表的结果，和100毫克/公斤N-乙基-N-亚硝基脲（ENU），后跟一个70天采样时间。这70天期间允许发生在精子的是精原干细胞在曝光时突变事件的测量（ 图3）。对突变体和滴定板典型斑块密度示于图7中所描绘于表2和图6，急性ENU曝光诱导显著剂量依赖性增加，精原干细胞的MF。低剂量诱导显著2.6倍，增幅比控制，其中有2.6×10 -5基线MF。最大感应发生在高剂量，这引起了控制为4.4倍的增加。 于生精小管精细胞进行该测定法的一个例子可以在道格拉斯等人 27，其中MF在以下5天重复腹膜内注射50毫克/公斤ENU不同的时间间隔来确定小管的生殖细胞中找到。在这项研究中，在生精细胞中的突变频率增加了15天的治疗后保持不变之后。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr tr=""><td> 世博会确定方案</td><td> 采集的组织 </td><td> 收集的细胞类型（靶细胞） </td><td> 靶细胞中的曝光的开始阶段 </td><td> 在曝光过程中通过阶段 </td></tr><tr><td>急性+70 </td><td>马尾</td><td>成熟精子</td><td>干细胞</td><td>干细胞</td></tr><tr><td> 14 + 21 </td><td>马尾</td><td>成熟精子</td><td>精原细胞</td><td>精子</td></tr><tr><td> 14 + 35 </td><td>马尾</td><td>成熟精子</td><td>干细胞</td><td>精母细胞</td></tr><tr><td> 28 +3 </td><td>马尾</td><td>成熟精子</td><td>精母细胞</td><td>精母细胞 精子 成熟精子</td></tr><tr><td></td><td>小管</td><td>干细胞</td><td>干细胞</td><td>干细胞</td></tr><tr><td></td><td></td><td>精原细胞</td><td> SPermatogonia </td><td>精原细胞</td></tr><tr><td></td><td></td><td>精母细胞</td><td></td><td>精母细胞</td></tr><tr><td></td><td></td><td>精子</td><td></td><td>精子</td></tr><tr><td> 28 + 49 </td><td>马尾</td><td>成熟精子</td><td>干CEL </td><td>干细胞</td></tr><tr><td></td><td>小管</td><td>干细胞 精原细胞 精母细胞 精子</td><td>干细胞</td><td>干细胞</td></tr></tbody></table> 这是由不同的实验设计，转基因啮齿动物突变试验针对表1细胞类型和精子的阶段。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 剂量组 </td><td> 动物＃ </td><td> 突变PFU </td><td> 总PFU </td><td> MF（X10-5） </td><td> 平均MF（×10 -5） </td><td> 倍数变化 </td><td> p值 </td></tr><tr><td>控制</td><td> 1 </td><td> 5 </td><td> 180245 </td><td> 2.8 </td><td> 2.6 </td><td> 1.0 </td><td> - </td></tr><tr><td></td><td> 2 </td><td> 4 </td><td> 137835 </td><td> 2.9 </td><td></td><td></td><td></td></tr><tr><td></td><td> 3 </td><td> 11 </td><td> 385672 </td><td> 2.9 </td><td></td><td></td><td></td></tr><tr><td></td><td> 4 </td><td> 2 </td><td> 431396 </td><td> 0.5 </td><td></td><td></td><td></td></tr><tr><td></td><td> 5 </td><td> 6 </td><td> 152413 </td><td> 3.9 </td><td></td><td></td><td></td></tr><tr><td> 25毫克/千克</td><td> 6 </td><td> 17 </td><td> 162353 </td><td> 10.5 </td><td> 6.9 </td><td> 2.6 </td><td> 0.0002 </td></tr><tr><td></td><td> 7 </td><td> 14 </td><td> 150094 </td><td> 9.3 </td><td></td><td></td><td></td></tr><tr><td></td><td> 8 </td><td> 4 </td><td> 154401 </td><td> 2.6 </td><td></td><td></td><td></td></tr><tr><td></td><td> 9 </td><td> 9 </td><td> 154401 </td><td> 5.8 </td><td></td><td></td><td></td></tr><tr><td></td><td> 10 </td><td> 12 </td><td> 196978 </td><td> 6.1 </td><td></td><td></td><td></td></tr><tr><td> 50毫克/千克</td><td> 11 </td><td> 17 </td><td> 155727 </td><td> 10.9 </td><td> 9.3 </td><td> 3.6 </td><td> &lt;0.0001 </td></tr><tr><td></td><td> 12 </td><td> 11 </td><td> 135847 </td><td> 8.1 </td><td></td><td></td><td></td></tr><tr><td></td><td> 13 </td><td> 25 </td><td> 193499 </td><td> 12.9 </td><td></td><td></td><td></td></tr><tr><td></td><td> 14 </td><td> 12 </td><td> 133859 &lt;/ TD&gt; <td> 9.0 </td><td></td><td></td><td></td></tr><tr><td></td><td> 15 </td><td> 14 </td><td> 252807 </td><td> 5.5 </td><td></td><td></td><td></td></tr><tr><td> 100毫克/公斤</td><td> 16 </td><td> 26 </td><td> 170968 </td><td> 15.2 </td><td> 11.5 </td><td> 4.4 </td><td> &lt;0.0001 </td></tr><tr><td></td><td> 17 </td><td> 28 </td><td> 234584 </td><td> 11.9 </td><td></td><td></td><td></td></tr><tr><td></td><td> 18 </td><td> 10 </td><td> 145289 </td><td> 6.9 </td><td></td><td></td><td></td></tr><tr><td></td><td> 19 </td><td> 35 </td><td> 292236 </td><td> 12.0 </td><td></td><td></td><td></td></tr><tr><td></td><td> 20 </td><td> 22 </td><td> 190848 </td><td> 11.5 </td><td></td><td></td><td></td></tr></tbody></table> 表2中的转基因雄性米马尾精子的lacZ基因的突变频率冰急性暴露于ENU当他们精原干细胞（管理时间为每日1次，采样时间为70天）。 PFU =空斑形成单位，MF =突变的频率。 <img alt="图1" fo:content-width="6in" width="600" src="/files/ftp_upload/51576/51576fig1highres.jpg" /> 图1中的λgt10噬菌体构建体的示意图。 <img alt="图2" fo:content-width="6in" width="600" src="/files/ftp_upload/51576/51576fig2highres.jpg" /> 图2转基因啮齿动物的生殖细胞突变试验的概况。 <img alt="图3" fo:content-width="6in" width="600" src="/files/ftp_upload/51576/51576fig3highres.jpg" /> 图3精子在小鼠和细胞类型和PHAS的示意图ES是由各种实验设计的转基因啮齿动物突变试验的目标。注：毕业白条代表存在于曲细精管细胞制成悬浮液的细胞类型（ 即精子&gt;精&gt;精原细胞&gt;干细胞）的相对比例。 <a href="https://www.jove.com/files/ftp_upload/51576/51576fig3highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图4" fo:content-width="6in" width="600" src="/files/ftp_upload/51576/51576fig4highres.jpg" /> 图4系列鼠标马尾附睾。 a）作出的对阴囊。 乙腹部的切口）确定附睾脂肪垫。 三）轻轻拉出脂肪垫绘制出睾丸和附睾。 四）找到并切除马尾附睾。 图由曲细精管5准备暂停生殖细胞中。 a）一个切口是在睾丸上皮胶囊制成，露出了曲细精管，B）中的曲细精管被挤出囊中。C）组织滚筒轻轻越过小管在5-10°角释放控制。ð内的生殖细胞）的胚细胞悬浮液被收集用于进一步处理。 <img alt="图6" fo:content-width="6in" width="600" src="/files/ftp_upload/51576/51576fig6highres.jpg" /> 的lacZ基因的突变频率在MutaMouse精子图6的图 ​​形表示急性暴露成体干细胞，以ENU组（n = 5），每个三角形代表一个动物的MF。* P &lt;0.05为泊松回归来确定。 <img alt="图7" fo:content-width="6in" width="600" src="/files/ftp_upload/51576/51576fig7highres.jpg" /> 形成于E的草坪图7代表琼脂平板上，从转基因啮齿动物突变试验与斑块大肠杆菌宿主菌。 A）中的 “突变体”板将有很少的斑块（用箭头标记），特别是在控制剂量组中，这有时会具有零斑块上的一些板。B）中的“效价”板可以有数百个噬菌斑。

Watch this Scientific Journal Video about Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency at JoVE.com

Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency

De novo mutations in the male germline may contribute to adverse health outcomes in subsequent generations. Here we describe a protocol for the use of a transgenic rodent model for quantifying mutations in male germ cells induced by environmental agents.

转基因检测鼠类量化男性生殖细胞突变频率

De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for ...

transgenic-rodent-assay-for-quantifying-male-germ-cell-mutant

Research

JoVE Journal

Biology

16.1K 次观看.  Environmental Health Centre. De novo mutations in the male germline may contribute to adverse health outcomes in subsequent generations. Here we describe a protocol for the use of a transgenic rodent model for quantifying mutations in male germ cells induced by environmental agents.

视频: Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

小学游离中脑多巴胺鼠类新生儿的细胞培养

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

科研

生物学

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

基因小鼠胚胎干细胞的基础含量为心源性诱导效率量化

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis1. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample2, 3. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. 
Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

量化传播肿瘤细胞的频率使用斑马鱼的同源有限稀释细胞移植

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal2. The use of donor males carrying the bacterial &beta;-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals1. Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located3. It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation4,5, to study Sertoli cell-germ cell interaction6,7, SSC homing and colonization3,8, as well as SSC self-renewal and differentiation9,10.
Germ cell transplantation is also feasible in large species11. In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species12. An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm13. Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice14.

Protocols for germ cell transplantation and testis tissue xenografting are described. Germ cell transplantation results in donor-derived spermatogenesis in recipient testes and represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs). Testis tissue xenografting reproduces testis development and spermatogenesis of various donor species in recipient mice.

生殖细胞的移植和睾丸组织在小鼠异种移植

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies ...

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell migration, gene expression, cell cycle progression and cell adhesion 1. Rho proteins are molecular switches that are active in GTP-bound and inactive in GDP-bound state. Their activation is mediated by a family of Guanine-nucleotide Exchange Factor (GEF) proteins. Rho-GEFs constitute a large family, with overlapping specificities 2. Although a lot of progress has been made in identifying the GEFs activated by specific signals, there are still many questions remaining regarding the pathway-specific regulation of these proteins. The number of Rho-GEFs exceeds 70, and each cell expresses more than one GEF protein. In addition, many of these proteins activate not only Rho, but other members of the family, contributing further to the complexity of the regulatory networks. Importantly, exploring how GEFs are regulated requires a method to follow the active pool of individual GEFs in cells activated by different stimuli. Here we provide a step-by-step protocol for a method used to assess and quantify the available active Rho-specific GEFs using an affinity precipitation assay. This assay was developed a few years ago in the Burridge lab 3,4 and we have used it in kidney tubular cell lines 5,6,7. The assay takes advantage of a "nucleotide free" mutant RhoA, with a high affinity for active GEFs. The mutation (G17A) renders the protein unable to bind GDP or GTP and this state mimics the intermediate state that is bound to the GEF. A GST-tagged version of this mutant protein is expressed and purified from E. coli, bound to glutathione sepharose beads and used to precipitate active GEFs from lysates of untreated and stimulated cells. As most GEFs are activated via posttranslational modifications or release from inhibitory bindings, their active state is preserved in cell lysates, and they can be detected by this assay8. Captured proteins can be probed for known GEFs by detection with specific antibodies using Western blotting, or analyzed by Mass Spectrometry to identify unknown GEFs activated by certain stimuli.

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein GST-RhoAG17A from Epithelial Cell Lysates

The method presented here describes an assay to follow activation of RhoA specific GDP/GTP Exchange Factors (GEFs) in cultured cells by making use of a mutant RhoA GST fusion protein that has high affinity for activated GEFs. GEFs are precipitated from cell lysates, detected by Western blotting and quantified by densitometry.

亲和沉淀主动RHO上皮细胞裂解液中使用的GST标签的突变Rho蛋白（GST-RhoA蛋白（G17A））全环基金

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell ...

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 108 cells/spermatogenic cell type from a starting population of 7-8 x 108 cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

生精细胞类型使用STA把速度沉降分离

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin&#8212;the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

解剖，成像和药物治疗： 果蝇蛹睾丸和单身男性生殖系囊肿的体外培养

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.

转基因的产生<em&gt;水润</em&gt;通过胚胎显微注射

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

可筛选的<em&gt;在体内</em&gt;测定线粒体调节剂使用转基因生物发光<em&gt;秀丽隐杆线虫</em

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

中通量筛选法评估对精子中 ca2 +信号和反应的影响

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...