这项工作是由动物和鸟类科学系到BT的资金支持。

所有动物实验均按照美国农业部的动物护理和使用指南批准贝尔茨维尔区动物护理和使用Comittees（BAACUC 11-015）。 1。麻醉和镇痛（常见的两种手术方法） <ol><li> 权衡鼠标和加载以下麻醉药和镇 ​​痛两个1毫升注射器27G的针头： <ol><li>氯胺酮（0.1毫克/克0.01毫升/克的10毫克/毫升的溶液）和甲苯噻嗪（0.01毫克/克0.005毫升/克的2毫克/毫升溶液）。 </li><li>丁丙诺啡（0.1微克/克0.01毫升0.01毫克/毫升的溶液）。 </li></ol></li><li>拿起它的脖子颈背尽量靠近钳口尽可能用拇指和食指和抱着小和无名指之间的尾部固定鼠标。 </li><li>注射氯胺酮-甲苯噻嗪混合腹腔 。为了避免刺穿内脏，按住鼠标其头部稍低于其臀部的水平（ 图1A） </li><li> 丁丙诺啡注射在皮下拇指和食指（ 图1B）之间保持脖子的颈背。 </li><li>离开鼠标在笼子里（干净，没有任何其他动物）在一个温暖的舞台。 </li><li>一旦昏迷，检查没有后脚反射 （由脚趾捏选中）。适用眼膏 ，以避免眼睛干涩，并核实没有眼睑反射消失 （ 图1C）。 </li><li>这个协议提供了一种外科麻醉平面为至少30分钟，足以执行下述程序（协议2和3）。如果需要更长的时间是必需的，额外的注射氯胺酮+甲苯噻嗪有一半在1.1.1中描述的剂量，可30分钟后应用。在呼吸模式，以更快的和不规则之一的变化表明LOSS适当的麻醉平面。 </li></ol> 2。输精管结扎术<ol><li>使用男用具有公认的插拔性能优良 。 </li><li> 消毒手术器械，其中清洁手术将要执行的曲面和用70％乙醇擦拭。 </li><li>如前面介绍（方案1），检查反射消失进行麻醉 。 </li><li>把鼠标放在一个温暖的阶段， 除去毛皮从两个假想横向线之间的腹侧区域电推子置于0.5厘米和2.5厘米以上的阴茎（ 图2A）。 </li><li>通过顺序擦拭，用10％聚维酮碘和70％乙醇消毒剃区域。 </li><li>鼠标放置在仰卧位，其对外科医生和盖用消毒毛巾用孔，露出剃区域尾巴。照亮手术区域。 </li><li>执行10-15毫米的纵向皮肤incisioN的中间线腹部，长约1厘米以上的阴茎。保持皮肤敷料锯齿钳，然后用剪刀剪（ 图2A和2B）。 </li><li> 在白线进行5-10毫米纵形切口 。保持肌肉与microdissecting锯齿钳和切割用剪刀（ 图2C）。 </li><li>抢一侧睾丸脂肪垫微解剖锯齿钳，将其暴露睾丸， 输精管和附睾 ， 输精管位于内侧的睾丸，这是一个清晰可辨的免费管（未连接到睾丸墙壁，好像附睾）与血管沿一侧（ 图2D）上运行。 </li><li>持输精管用微解剖锯齿钳， 火焰敷料钳，直到它们变成红色（ 图2E </st荣&gt;），然后用它们在一次切割和烧灼输精管的两个点（ 图2F）。切口应除去约5毫米的部分，留下两个明显分开的烧灼结束（ 图2G）。 </li><li>将睾丸，附睾和输精管回腹腔。 </li><li> 从其他睾丸第9步进行 。 </li><li> 缝合肌肉与5/0可吸收缝线（ 图2H）做一个或两个水平褥式缝合。 </li><li> 缝合与一个或两个伤口剪（ 图2I）皮肤 。 </li><li>识别vasectomised男性（耳环，手指纹身...），将它的笼子放置在一个温暖的阶段， 观察，直到它从麻醉中恢复 （意识和维护胸骨斜卧）。一个0.5-1毫升皮下注射温生理盐水溶液再提高covery。输精管结扎记录在传输过程中发生的可能发生率，抗生素添加到饮用水。 </li><li>卷绕剪辑可以输精管切除术与伤口剪刀去除或一对齿钳10天之后被去除 。在男性输精管结扎会准备交配术后2周。 </li><li>通过使用它来 ​​获取收件人之前，肥沃的女性交配试验的输精管结扎男性的不育 。 </li></ol> 3。子宫 - 输卵管胚胎移植<ol><li>小鼠桑椹胚或囊胚可以通过这种技术被转移到假孕雌性接受者在2.5 DPC。 </li><li>准备操纵胚胎玻璃吸管 ： <ol><li>研磨玻璃毛细管的尖端，以避免损坏吸液管保持。 </li><li>通过加热用细火而略微旋转软化的玻璃毛细管的中间部分毛细管用双手同步。一旦毛细管部分变得柔软，延展性（淡红色），迅速 ​​从火焰撤回拉两端，其外部直径缩小到130-150微米。 </li><li>等待玻璃冷却下来，然后剪轻轻得分狭窄部分用钻石点铅笔，研磨石或指甲锉，并从两侧拉。休息应该是干净和垂直</li></ol></li><li> 抛光前部，通过非常迅速燃烧，留下了100-130微米的孔径。移液器可以存储供以后使用。 </li><li>温暖的胚胎操纵媒体 （CZBH或M2，见讨论）。 </li><li> 消毒手术器械，其中清洁手术将要执行的曲面和用70％乙醇擦拭。 </li><li>如前面介绍（方案1），检查反射消失进行麻醉 。 </li><li>保持T他的鼠标在一个温暖的阶段， 除去毛皮从膝盖和远端肋骨（ 图3A和3B）之间的背侧区电推剪。 </li><li>通过顺序擦拭，用10％聚维酮碘和70％乙醇消毒剃区域。 </li><li> 将胚胎从孵化器到预热的胚胎操纵媒体。 </li><li>将收件人温暖的阶段，体视显微镜下，并将其放置在俯卧位横向外科医生（与它的头向右看或外科医生的左侧）。 </li><li>用无菌毛巾用孔，露出剃区域覆盖的区域，照亮手术区域。 </li><li>执行1厘米的横向（垂直） 切口，在皮肤中位于最后肋骨和臀部和背部和腹部（ 图3A中的线之间的背⅓之间的线的颅⅓一个点ND 3B）。持与敷料锯齿钳子皮肤和用剪刀剪（ 图3C）。 </li><li>一旦皮肤已被切断，卵巢（红/橙）或周围的卵巢（白色）的脂肪垫可以通过体壁进行可视化。 在体壁进行0.3-0.5厘米的横向（垂直）的切口比在一个地方的切口不切断任何大血管的卵巢或脂肪垫。持与微解剖锯齿钳子肌肉和用剪刀剪下（ 图3D）。 </li><li>将鼠标移动到具有其头部朝向外科医生。 </li><li> 加载胚胎操纵吸管 （ 图3E）： <ol><li>允许CZBH介质通过毛细管现象通过操纵移液管的窄部提升至较宽的部分为约5毫米。 </li><li>拿起一个小气泡（0.2-0.5毫米）。 </li><li>在引进胚胎（5-10）媒体少量（2-4毫米）。 </li><li>拿起另一个小气泡（0.2-0.5毫米）和少量的媒体（0.5-1毫米）。 </li><li>留下连接到口吸气持有人或手动操作的装置，准备步骤3.17的玻璃移液管。 </li></ol></li><li>抓住周围用微解剖锯齿钳子卵巢脂肪垫并把它朝着小鼠头部暴露卵巢，输卵管，以及一小部分的上子宫出腹腔（ 图3F和3G）。 </li><li>握住吸气口片在口中，随时可以使用，抢脂肪垫微解剖锯齿钳将输卵管和暴露子宫-输卵管交界处（ 即其中输卵管达到子宫）。 </li><li>保持子宫 - 输卵管交界处前往，采取微弯微解剖钳用左手（如果右撇子），并把它们的正下方在子宫-输卵管交界处抓长约2毫米以上的部分输卵管 。 </li><li>握住子宫-输卵管交界处的微弯微解剖钳， 穿破输卵管部分接近钳用27号针头（ 图3H）。 </li><li> 将胚胎操纵吸管插入通过子宫-输卵管交界处用针和前进到子宫进行的小孔（ 图3I和3J）。一旦移液器已通过子宫-输卵管交界处（ 图3K），它的幻灯片轻松。不进步到很远的子宫，防止子宫内膜损伤（不超过3毫米）和吸管堵塞的杂物。 </li><li>轻轻吹（ 图3L）释放的胚胎进入子宫。这两个气泡必须通过子宫。一些媒体的第一泡以上也可以释放到子宫，但避免引入更多空气，因为它可能会妨碍着床。 </li><li> 除去胚胎已被释放到子宫刚过吸管 。 </li><li>将输卵管和抓取脂肪垫子房回腹腔。 </li><li>用5/0可吸收缝线（ 图3M）缝合肌肉与水平褥式缝合。 </li><li> 缝合伤口剪（ 图3N）的皮肤 。 </li><li> 如果需要从另一侧步骤10继续。 </li><li>识别收件人（耳环，手指纹身...），将其移动到它的笼子（放置在一个温暖的阶段），然后观察，直到它从麻醉中恢复 （意识和维护胸骨斜卧）。 </li><li>注释胚胎移植过程中发生的可能发生率，并添加抗生素的饮用水。一个0.5-1毫升皮下注射温暖萨利的NE解决方案改善恢复。 </li><li>伤口剪辑可以胚胎移植的伤口剪去除剂或两对钳（ 图1-30）后10天被删除 。收件人可以在这一天来评估怀孕和估计幼仔的数量来衡量。胚胎移植后15天偎依提供材料给收件人。 </li></ol>

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作者宣称没有竞争的财务权益。

输精管结扎术是一种相对简单的手术技术，不涉及重大困难。当用聚维酮碘和乙醇消毒确保最后一次洗涤（用乙醇）去除碘伏，因为它会刺激腹膜。到输精管的访问也可以通过阴囊或在腹部8进行横向切口实现的。阴囊切口已被推荐到腹部横切口，由于需要相对较小的切口，并稍微好一点的术后行为11。然而，我们优选腹部切口在阴囊，因为它提供了从两个睾丸到输精管一种更简单和更清楚的访问，从而防止新手外科医生从烧灼两次相同的输精管 ，留下一个官能输精管 。腹部两者之间的技术，我们看好的纵向切口，在白线在颠SAL，因为它不切割任何腹部肌肉纤维，避免腹部疝的发展。但是，这两个输精管切除术和子宫 - 输卵管胚胎传输协议可以适应可用的设备或研究者的个人喜好。例如，玻璃珠消毒器可用于加热用于烧灼输精管的钳子。在任何情况下，必须遵循无菌技术，为客户提供适当的麻醉和镇痛，以最大限度地提高动物福利，并遵守当地法规。 另一方面容易的修改是麻醉协议。如果吸入麻醉（异氟烷）可用，我们鼓励其使用，而不是胃肠道（注射）麻醉，因为它提供了一个非常稳定的麻醉平面和快速恢复。然而，重要的是要指出，吸入麻醉仍然要求使用镇痛剂的区域内和后手术室疼痛，这应该是政府上台术前用药为之前来选取。丁丙诺啡是一种阿片样物质，提供在鼠标12长期镇痛。肠外麻醉也提供了良好的麻醉平面短程序，如输精管结扎术和胚胎移植。氯胺酮-甲苯噻嗪对小鼠手术13一个非常可靠的结合，并与术前给予丁丙诺啡一起使用这个组合，我们从来没有观察到任何麻醉并发症。其它胃肠组合可用于12，但我们不鼓励阿佛丁（三溴乙醇）使用麻醉剂的混合物，因为只有一个单一的剂量可管理和多篇文章报道其使用，如局部刺激，镇痛差，肠梗阻相关的并发症多样，在腹腔内，腹膜下的肌肉纤维和腹部器官表面坏死，甚至死亡14-18纤维性粘连。 胚胎移植既需要胚胎良好的管理ð收件人。作为用于哺乳动物胚胎移植一般规则，在胚胎的发育阶段可以比接收方的假孕阶段更先进，但是反之则不行。换句话说，胚胎可以等待母亲，但母亲不能等待胚胎，所以这种技术可以用来传输桑椹胚或囊胚，但不是早期阶段。子宫 - 输卵管胚胎移植可两天后检测阴道塞从中午（2.5 DPC）到傍晚晚（3 DPC），当子宫 - 输卵管交界处，是开放的，允许胚胎的自然中转从输卵管进行子宫角。考虑到转移的胚胎可能已经从某种操作的遭遇，胚胎的处理应尽量减少任何进一步的损害。对小鼠胚胎操纵一个很好的指南可以在纳吉等人发现。8两种最常用的小鼠胚胎操纵媒体，这在常规举行的生理pH值大气，是CZBH 19和M2 20。虽然在体内产生的小鼠胚胎可以克服暴露在寒冷温度或pH值异常长时间21，操纵媒体应预热。如果介质预热在培养皿中，应避免长时间（超过40分钟）升温，作为媒体的渗透压摩尔浓度会增加，由于水分蒸发，而且可以比冷操纵媒体更有害。类似地，将胚胎操纵吸管内所花费的时间应该是由于小体积中存在的移液管最小化。 使用适当的胚胎操纵吸管的是该协议的成功是至关重要的。操作移液器可以从玻璃毛细管薄薄的玻璃墙进行。巴斯德移液管，经常被用来处理大量的动物胚胎，也可以使用，但由于从手柄的短距离之内的前端，由玻璃制成的毛细管吸液管操作更COMFortable操纵。暴露于拉动火焰和速度决定了壁的厚度和吸移管的内径。虽然操纵吸管可以用手很容易地完成，也可以使用一些实践中，拔出器和微锻造后。重要的是要投入时间生产几个最佳操作移液管是很重要的。操纵吸管孔径应大于胚胎更宽，以允许平稳流动，但是足够小以容易地渗透并穿过子宫 - 输卵管交界进步。如果透明带已被移交前拆除，囊胚通常扩展到更大的直径。在这种情况下，最好是使用较大的光圈（130-180微米）更宽的移液管，以避免对滋养层细胞的任何损害。尖抛光是很重要的，以避免损坏输卵管，子宫壁和胚胎-特别是如果透明带已被去除，并避免移液管尖被阻止碎屑。然而，在抛光后，孔径应该Ñ加时过小的移液管的内径相比较，如在直径急剧减少将会导致流动速度的突然变化。抽吸嘴件提供了更精确的流量控制，以及相对于手控装置更舒适的手的位置。然而，如果需要的话（例如操纵慢病毒处理的胚胎时），可以使用一只手控制的设备。为获得最佳流动，并避免矿物油的转移，也建议使用一种新的移液管用于胚胎移植的而不是用于移动从培养介质中的胚胎操纵媒体。最后，虽然通过输卵管引入移液器，毛细管应直接处理， 即得 。抓住了玻璃，而不是吸引的塑料手柄，以获得牢固的抓地力。用于胚胎移植的放大倍数是个人的事，它取决于外科医生的视力。我们优选使用低倍率为具有宽视场，所以我们用10倍的放大倍率决赛（10X目镜和1X物镜）。 收件人应至少8周龄之间27-40克的重量。远系繁殖的小鼠如CD1或瑞士韦伯斯特显示了良好的母性行为和优良收件人。明智的做法是成立养殖获得额外的收件人，那些不使用将恢复其正常的循环活动两个星期。尽管被配合时，一些女性可能没有黄体（ 图3G），并因此将不容易接受移植胚胎。出于这个原因，卵巢应检查黄体，这在2.5 DPC可以明确认定为鲜红色结构在卵巢（ 图3F）的存在。过度操纵的卵巢和子宫，可能会导致黄体，在手术过程中它通过抓取卵巢脂肪垫，而不是尽量减少与生殖道的接触是很重要的。该manipulatio引进Ñ​​吸管进入输卵管是协议中最复杂的一步。在一些接收者，输卵管是非常扭曲，并且没有一个2毫米的直伸展从与子宫的交界处。在这种情况下安排的输卵管和在所述第一弯曲部进行穿刺。如上文所详述，一个不错的操控吸管真的有差别。一旦移液器已通过子宫 - 输卵管交界处通过，容易滑动。如果不是这样，该吸液管可能已经从输卵管偏离并没有到达子宫腔。子宫一旦进入，如果媒体不流动，使移液管稍微或并再试一次。如果仍然不流，移液管堵塞，把它从子宫，以操纵媒体发布的菜的内容并重新加载相同或其他移液管。送货通常发生胚胎移植后17天。为了防止自相残杀，提供偎依材料提前2天，并在分娩后的头几天不改变笼。

胚胎移植可能是在小鼠模型中进行的最常见的外科手术。这种技术是必不可少的，从胚胎进行体外操作技术获得的后代，因此，构成了转基因模型中通过原核注射，慢病毒转导，或嵌合体的形成的发展的必要步骤。此外，该技术允许的早期发育过程中出现的多种有害刺激的发育影响的研究。使用人工繁殖技术1或接触到不同的物质或代谢物2浓度异常可能会影响导致植入或胎盘失败和后代的长期影响胚胎发育。一个可靠和可重复的胚胎移植技术是至关重要的测试实验处理对着床和胎儿发育一致的人可能产生的负面影响NER。 小鼠植入前胚胎可以通过的0.5天交配后壶腹（DPC）假收件人（输卵管转让）3,4或成2.5 DPC假收件人的子宫（子宫转让）5,6传送到收件人的女性要么进入输卵管根据它们的发育阶段。胚胎在胚泡期，如那些用于产生嵌合小鼠通过注射胚胎或诱导多能干细胞的，通常是由子宫转移转移。囊胚也可以转移到0.5 DPC收件人的输卵管，但它构成了一个不太生理测试发育干扰，因为胚胎滞育经历，并具有2天的侮辱恢复植入前发生。子宫转让涉及的穿刺子宫壁具有窄的针，以便产生一个孔，它允许一个胚胎操纵吸管的访问到子宫腔中。一lthough这种技术可以产生良好的效果，生存至足月（ 即移植胚胎的发育到小狗的百分比）往往是低和难以预料7,8。 子宫壁的穿刺则需要进行一些有害的副作用。首先，子宫肌层是高度血管化组织和其穿刺通常导致一个小的出血。血液可能会阻止胚胎移液管或侵入子宫腔引起胚胎死亡和/或植入失败。这是特别相关的，当胚卵不被转移，如血细胞和碎片可以连接到卵裂球。第二，所执行的开口不密封的胚胎已转移后，使他们能够流回通过节流孔和排出到腹腔时，过大的体积已经被引入到子宫。本文所述的子宫 - 输卵管胚胎移植采取子宫 - 输卵管交界处的优势为客户提供电磁制动YOS进入子宫而不刺穿子宫壁，从而避免其不利后果9的需要。 假孕雌性受体用于胚胎移植是通过自然交配获得与输精管结扎男性8。由雄性不育产生的精液分泌物都需要子宫成为容易接受移植胚胎。为了获得一个收件人，最多2女8周〜6个月的婴儿被​​放在了输精管结扎男性在下午。次日早晨，女性被检查阴道交配插头，从男性精液凝固蛋白的一丛的存在。随着午夜的交配过程中通常会发生，阴道塞检测的日子被认为是0.5 DPC。虽然男性输精管结扎可以从一些供应商购买，这里所描述的手术过程是比较容易的，不需要任何额外的工具不是必需的胚胎移植。

<table><tbody><tr><td>Ketamine</td><td>VEDCO</td><td>Ketaved ANADA 200-257</td><td>To be ordered by a licensed veterinarian.</td></tr><tr><td>Xylazine</td><td>Lloyd Laboratories</td><td>Anased NADA #139-236</td><td>To be ordered by a licensed veterinarian.</td></tr><tr><td>Buprenorphine</td><td>Generic</td><td>NDC 400-42-010-01</td><td>To be ordered by a licensed veterinarian.</td></tr><tr><td>Eye ointment</td><td>Novartis</td><td>Genteal</td><td></td></tr><tr><td>Antibiotic</td><td>Pfizer</td><td>Clavamox NADA #55-101.</td><td>Added to drinking water (0.3 mg/ml of amoxicillin trihydrate and 0.075 mg/ml of clavulanate potassium). Add 1.5 ml of the reconstituted 15 ml bottle to 250 ml of water.</td></tr><tr><td>Dressing serrated forceps</td><td>ROBOZ</td><td>RS-8120</td><td>Any medium size surgical-grade steel straight forceps will work.</td></tr><tr><td>Microdissecting serrated forceps</td><td>ROBOZ</td><td>RS-5137</td><td>These ones are curved at a 90&amp;ordm; angle. Straight forceps can be used if preferred.</td></tr><tr><td>Slight curved micro dissection forceps</td><td>ROBOZ</td><td>RS-5136</td><td>This model is particularly useful to hold the oviduct.</td></tr><tr><td>Scissors</td><td>ROBOZ</td><td>RS-5880</td><td>Any regular surgical grade steel small straight scissors will work.</td></tr><tr><td>27 G needles</td><td>Beckton-Dickinson</td><td>305136</td><td>Smaller needles (30 G) can be also used. 25 G may be a bit too big.</td></tr><tr><td>Clip applier</td><td>MiKRon</td><td>42763</td><td></td></tr><tr><td>9 mm Clips</td><td>MiKRon</td><td>427631</td><td></td></tr><tr><td>Clip remover</td><td>MiKRon</td><td>7637</td><td>Two pairs of teeth forceps (ROBOZ RS-8160) can be used instead.</td></tr><tr><td>Suture needle holder</td><td>ROBOZ</td><td>RS-7820</td><td></td></tr><tr><td>Suture</td><td>Dowist Gell</td><td>5-0 Dexon S 7204-21</td><td>Can be substituted for any 4-0 to 6-0 absorbable suture with a narrow curved needle.</td></tr><tr><td>Glass capillaries</td><td>VWR</td><td>100 ul calibrated pipettes 53432-921</td><td>It includes a mouth aspirator system that only requires to attach a 0.22 &amp;#181;m filter in the tubing to be ready to use. More information can be obtained in Nagy &lt;em&gt;et al&lt;/em&gt;.&lt;sup&gt;8&lt;/sup&gt;</td></tr><tr><td>Burner</td><td>KISAG AG</td><td>Typ 2002</td><td>Gas operated burner, can be charged with Kigas (CH-4512, from the same vendor). Alcohol burners may be also used, but gas provides a higher temperature and this burner provides a small and precise flame.</td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>MZFLIII</td><td>This is an expensive estereomicroscope with fluorescence, that can be also used for other purposes. There are cheaper options such as Leica MZ8 or Nikon SMZ-10 or SMZ-2B, to name a few. It is better to use two stereomicroscopes, one for handling the embryos (which does not need to be a very nice one) and another one for the recipient. The one used for the recipient should display a long distance from the stage plate to the objective lense, in order to be able to focus 3-4 cm above the stage plate (where the oviduct will be placed) and still leave some room for the surgeon; most of the stereomicroscopes can do this, but some cannot.&amp;nbsp;</td></tr><tr><td>Fiber optics ilumination</td><td>Dolan Jenner</td><td>Fiber lite</td><td>To iluminate the surgical area. There are different systems available.</td></tr><tr><td>Warm stages</td><td>American scope</td><td>&lt;a href=&quot;https://exch.mail.umd.edu/owa/redir.aspx?C=1qTJKejamUSfudeYG_MlZHvTY3dsJM9Iaqr2SnwGD4ECWGzB8e8Rpg--_L7u0vJmyatXQJzGQ0k.&amp;amp;URL=http%3a%2f%2fstore.amscope.com%2ftcs-100.html&quot; target=&quot;_blank&quot;&gt;http://store.amscope.com/tcs-100.html&lt;/a&gt;</td><td>These can be placed over the stage plate of the stereomicroscope. Some modifications (inserting a stick to level the stage) may be needed if it is too short for the stereomicroscope. A big warm stage can be used for warming the cage if it is available. If not, a regular heating pad can be used, but temperature must be checked.</td></tr><tr><td>Culture dishes for embryo manipulation</td><td>Falcon</td><td>353001</td><td>351008 may be also used; they made narrower drops.</td></tr></tbody></table>

utero-tubal embryo transfer and vasectomy in the mouse model

植入前胚胎到代孕雌鼠转移是用于生产转基因小鼠或研究遗传学改变的影响源自于随后的胚胎发育和成年人的健康胚胎植入前发育过程中的必要步骤。使用有效的和一致的胚胎移植技术的关键是要提高转基因动物的产生和确定不同处理对植入率和存活期的影响。胚胎在囊胚阶段通常是由子宫转移转移，在子宫壁进行穿刺引进胚胎操纵吸管。在子宫中执行的孔口不会关闭吸管已经撤回后，与胚胎可以流出到腹腔由于子宫的正压力。穿刺也可产生出血，损害植入，块的移液管，并可能影响胚胎ð才有发展，尤其是当胚胎没有透明传送。因此，这种技术通常会导致非常多变和整体低胚胎存活率。避免这些负面影响，子宫 - 输卵管胚胎移植采取子宫 - 输卵管交界处，阻碍胚胎流出一道天然屏障的优势，避免了子宫壁的穿刺。输精管结扎的男性都需要获得假收件人。的技术进行输精管结扎术被描述为补充的子宫 - 输卵管胚胎移植。

子宫-输卵管胚胎移植提供了一个刻薄的胚胎转移到子宫，避免了一些关联到子宫胚胎移植2,9,10并发症。在表1中我们将展示我们所获得把经受不同种类的操作，以CD1收件人CD1囊胚下面描述的协议的一些代表性的结果。的生存期（导致小狗胚胎％）或存活至E15（在慢病毒暴露的情况下）是胚胎只是在体外培养从受精卵阶段（IVC），胚胎经受IPSC的注射，以产生嵌合的幼仔之间相似和胚胎有其卵取出并暴露于慢病毒对胚胎移植前7小时。因此，子宫-输卵管胚胎移植是一种可靠的技术转移胚胎特别复杂，例如那些缺乏透明带和孵育慢病毒。 图1。注射麻醉剂和止痛剂A）腹腔注射氯胺酮-甲苯噻嗪B）皮下注射眼膏的丁丙诺啡，C）中的应用。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51214/51214fig2highres.jpg" src="/files/ftp_upload/51214/51214fig2.jpg" /> 图2。 。输精管结扎术协议A）的切口点（描绘一个红色的“X”）被放置约1厘米以上的阴茎;。从0.5-2.5厘米以上的阴茎（黑线）B）皮肤切口C）肌肉切口去除毛皮。 D） 输精管 （黑色箭头）和测试是（*）E）火红的镊子在两个点一次输精管烧灼F）烧灼G） 输精管清楚地分为两个烧灼结束。 高）肌肉缝合。 我 ）皮肤缝合。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51214/51214fig2highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51214/51214fig3highres.jpg" src="/files/ftp_upload/51214/51214fig3.jpg" /> 图3。子宫-输卵管胚胎移植协议A，B）和背侧切口点）侧面观（描绘一个红色的“X”;最后肋骨和臀部之间的黑线（A，B）和背部和腹部之间（B）作为指导。 三）皮肤切口。 <stroNG&gt; D）肌肉切开E）装有5囊胚准备转移胚胎操纵玻璃吸管，F，G）和（F）或无（G）黄体代表卵巢，用黑色箭头。 香港 ）为代表的目的，进行了模拟子宫-输卵管胚胎移植装入玻璃吸管操纵用0.4％台盼蓝溶液输卵管H）穿刺（黑色箭头）接近子宫（*）。 我 ）胚胎操纵吸管的引入孔以前执行J）胚胎操纵吸管要通过台盼相当于蓝色的溶液中释放出来的胚胎移植的卷已被开除在子宫腔（黑色箭头的子宫-输卵管交界K））。长）前进测试由子宫 - 输卵管交界处产生的有效封闭，所有的操纵吸管的内容被释放到子宫内，子宫-输卵管结（箭头）阻碍释放到子宫的台盼蓝溶液（*）M）肌肉缝合N）的皮肤缝合O）剪辑去除的背面流动。。 15天后P）的胚胎移植提供偎依材料给收件人。Q）以下这种技术获得的嵌合垃圾。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51214/51214fig3highres.jpg" target="_blank">点击这里查看大图。</a> <table border="0" cellpadding="0" cellspacing="0" width="583"><colgroup><col /><col /><col /><col /><col /></colgroup><tbody><tr height="20"><td height="20" style="height:20px;width:91px;">治疗</td><td style="width:135px;">传输数</td><td style="width:131px;">移植胚胎</td><td style="width:99px;">交付幼崽</td><td style="width:131px;">生存至tERM（％） </td></tr><tr height="20"><td height="20" style="height:20px;">下腔静脉</td><td align="center"> 11 </td><td align="center"> 110 </td><td align="center"> 82 </td><td align="center"> 74.5 </td></tr><tr height="20"><td height="20" style="height:20px;"> IPSC的注射</td><td align="center"> 3 </td><td align="center"> 50 </td><td align="center"> 35 </td><td align="center"> 70.0 </td></tr><tr height="20"><td height="20" style="height:20px;">慢病毒</td><td align="center"> 6 </td><td align="center"> 60 </td><td align="center"> 44 </td><td align="center"> 73.3 </td></tr></tbody></table> 表1中。转移三组不同的操纵胚胎以下子宫-输卵管胚胎移植后获得的代表性结果。1）胚胎体外培养（IVC）从受精卵阶段至胚泡期，2） 在注射10按钮鼠体内产生的胚泡Ë的iPSC生成嵌合体小鼠，3） 在体内产生的胚泡的有他们的卵取出，孵育7小时与表达GFP的慢病毒。数据代表诞生出转移除慢病毒暴露组，在那里它们代表可行的胎儿的数目胚胎移植后第10天，因为妊娠是不允许​​进一步进步胚胎数的幼仔的数量。

Watch this Scientific Journal Video about Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model at JoVE.com

Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

子宫 - 输卵管胚胎移植使用子宫 - 输卵管结作为一个屏障，以防止子宫进行传输时可能出现的胚胎流出。输精管结扎男性须取得假收件人胚胎移植。这两种技术都进行了讨论。

子宫 - 输卵管胚胎移植和输精管结扎术在小鼠模型

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects ...

utero-tubal-embryo-transfer-and-vasectomy-in-the-mouse-model

Universidad San Sebastian

Research

JoVE Journal

Biology

47.7K 次观看.  United States Department of Agriculture. 子宫 - 输卵管胚胎移植使用子宫 - 输卵管结作为一个屏障，以防止子宫进行传输时可能出现的胚胎流出。输精管结扎男性须取得假收件人胚胎移植。这两种技术都进行了讨论。 

视频: Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

一个小鼠模型在子宫内移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

科研

医学

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

超声引导下显微注射到小鼠前脑在子宫内在E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

神经科学

Mouse Model of Surgically-induced Endometriosis by Auto-transplantation of Uterine Tissue

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of lesions, and/or chronic pharmaceutical management of pain and infertility symptoms. The cost associated with endometriosis has been estimated at 22 billion dollars per year in the United States1. To further our understanding of mechanisms underlying this enigmatic disease, animal models have been employed. Primates spontaneously develop endometriosis and therefore primate models most closely resemble the disease in women. Rodent models, however, are more cost effective and readily available2. The model that we describe here involves an autologous transfer of uterine tissue to the intestinal mesentery (Figure 1) and was first developed in the rat3 and later transferred to the mouse4. The goal of the autologous rodent model of surgically-induced endometriosis is to mimic the disease in women. We and others have previously shown that the altered gene expression pattern observed in endometriotic lesions from mice or rats mirrors that observed in women with the disease5,6. One advantage of performing the surgery in the mouse is that the abundance of transgenic mouse strains available can aid researchers in determining the role of specific components important in the establishment and growth of endometriosis. An alternative model in which excised human endometrial fragments are introduced to the peritoneum of immunocompromised mice is also widely used but is limited by the lack of a normal immune system which is thought to be important in endometriosis2,7. Importantly, the mouse model of surgically induced endometriosis is a versatile model that has been used to study how the immune system8, hormones9,10 and environmental factors11,12 affect endometriosis as well as the effects of endometriosis on fertility13 and pain14.

A description of the surgical induction of endometriosis in mice and rats by auto-transplantation of uterine tissue to the arterial cascade of the intestinal mesentery.

自动子宫组织移植手术诱发子宫内膜异位症小鼠模型

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of ...

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.

Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice.

使用冷冻胚胎进行转基因小鼠高效生产

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. ...

遗传学

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment.

小鼠宫内移植中静脉和羊膜内植入的研究

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...

发育生物学

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

家兔模型最佳胚胎阶段微创胚胎移植与胚胎玻璃化

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model

For pregnancy to be established, a viable blastocyst must successfully interact with a receptive uterine lining (endometrium) to facilitate implantation and placenta formation and enable ongoing pregnancy. The limitations to pregnancy success caused by embryonic defects are well known and have been largely overcome in recent decades with the rise of in vitro fertilization (IVF) and assisted reproductive technologies. As yet, however, the field has not overcome the limitations caused by an inadequately receptive endometrium, thus resulting in stagnating IVF success rates. Ovarian and endometrial functions are closely intertwined, as hormones produced by the ovary are responsible for the endometrium's menstrual cyclicity. As such, when using rodent models of pregnancy, it can be difficult to ascertain whether an observed result is due to an ovarian or uterine deficit. To overcome this, an ovariectomized mouse model was developed with embryo transfer or artificial decidualization to allow the study of uterine-specific contributions to pregnancy. This article will provide instructions on how to perform ovariectomy and offer insights into various techniques for supplying exogenous hormones to support successful artificial decidualization or pregnancy following embryo transfer from healthy donors. These techniques include subcutaneous injection, slow-release pellets, and osmotic mini pumps. The key advantages and disadvantages of each method will be discussed, enabling researchers to choose the best study design for their specific research question.

Pregnancy establishment is a dynamic process involving complex embryo and uterine crosstalk. The precise contributions of the maternal uterine environment to these processes remain an active area of investigation. Here, detailed protocols are provided to aid in designing in vivo animal models to address these research questions.

研究卵巢切除小鼠模型中子宫对妊娠建立的贡献的方法

For pregnancy to be established, a viable blastocyst must successfully interact with a receptive uterine lining (endometrium) to facilitate implantation ...

<meta charset="utf8"></head><body>小鼠圆形精子细胞注射程序研究胚胎发育

Mouse Round Spermatid Injection

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of round spermatid injection (ROSI), oocytes can be successfully fertilized and developed into viable fetuses. In a groundbreaking milestone achieved in 1995, the first mouse fetus was born through ROSI technology. ROSI has since emerged as a pivotal tool for unraveling the intricate mechanisms governing embryonic development and holds significant potential in various applications, including the acceleration of mouse generation and the production of genetically modified mice. In 1996, a milestone was reached when the first human fetus was born through ROSI technology. However, the clinical applications of this method have shown a fluctuating pattern of success and failure. To date, ROSI technology has not found widespread application in clinical practice, primarily due to its low birth efficiency and insufficient validation of fetal safety. This article provides a comprehensive account of the precise methods of performing ROSI in mice, aiming to shed new light on basic research and its potential clinical applications.

<meta charset="utf8"></head><body>小鼠圆形精子细胞注射

Here, we introduce a method for performing round spermatid injection (ROSI) in mice, a technique with promising clinical applications and utility for investigating the mechanisms underlying embryonic development.

小鼠圆形精子细胞注射

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of ...

子宫 - 输卵管胚胎移植和输精管结扎术在小鼠模型

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一个小鼠模型在子宫内移植

超声引导下显微注射到小鼠前脑在子宫内在E9.5

自动子宫组织移植手术诱发子宫内膜异位症小鼠模型

使用冷冻胚胎进行转基因小鼠高效生产

小鼠宫内移植中静脉和羊膜内植入的研究

家兔模型最佳胚胎阶段微创胚胎移植与胚胎玻璃化

研究卵巢切除小鼠模型中子宫对妊娠建立的贡献的方法

小鼠圆形精子细胞注射

小鼠圆形精子细胞注射

小鼠圆形精子细胞注射