子宫 - 输卵管胚胎移植和输精管结扎术在小鼠模型
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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） 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： 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）。 <…
Representative Results
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…
Discussion
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</…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由动物和鸟类科学系到BT的资金支持。
Materials
| Ketamine | VEDCO | Ketaved ANADA 200-257 | To be ordered by a licensed veterinarian. |
| Xylazine | Lloyd laboratories | Anased NADA #139-236 | To be ordered by a licensed veterinarian. |
| Buprenorphine | Generic | NDC 400-42-010-01 | To be ordered by a licensed veterinarian. |
| Eye ointment | Novartis | Genteal | |
| Antibiotic | Pfizer | Clavamox NADA #55-101. | 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. |
| Dressing serrated forceps | ROBOZ | RS-8120 | Any medium size surgical-grade steel straight forceps will work. |
| Micro dissecting serrated forceps | ROBOZ | RS-5137 | These ones are curved at a 90º angle. Straight forceps can be used if preferred. |
| Slight curved micro dissection forceps | ROBOZ | RS-5136 | This model is particularly useful to hold the oviduct. |
| Scissors | ROBOZ | RS-5880 | Any regular surgical grade steel small straight scissors will work. |
| 27G needles | Beckton-Dickinson | 305136 | Smaller needles (30G) can be also used. 25G may be a bit too big. |
| Clip applier | MiKRon | 42763 | |
| 9 mm Clips | MiKRon | 427631 | |
| Clip remover | MiKRon | 7637 | Two pairs of teeth forceps (ROBOZ RS-8160) can be used instead. |
| Suture needle holder | ROBOZ | RS-7820 | |
| Suture | Dowist Gell | 5-0 Dexon S 7204-21 | Can be substituted for any 4-0 to 6-0 absorbable suture with a narrow curved needle. |
| Glass capillaries | VWR | 100 ul calibrated pipettes 53432-921 | It includes a mouth aspirator system that only requires to attach a 0.22 um filter in the tubing to be ready to use. More information can be obtained in ref. 8. |
| Burner | KISAG AG | Typ 2002 | 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. |
| Stereomicroscope | Leica | MZFLIII | 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. |
| Fiber optics ilumination | Dolan Jenner | Fiber lite | To iluminate the surgical area. There are different systems available. |
| Warm stages | American scope | http://store.amscope.com/tcs-100.html | 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. |
| Culture dishes for embryo manipulation | Falcon | 353001 | 351008 may be also used, they made narrower drops. |
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Cite This Article
Bermejo-Alvarez, P., Park, K., Telugu, B. P. Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model. J. Vis. Exp. (84), e51214, doi:10.3791/51214 (2014).