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>

<ol>
	<li>Fernandez-Gonzalez, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+effect+of+in+vitro+culture+of+mouse+embryos+with+serum+on+mRNA+expression+of+imprinting+genes,+development,+and+behavior.">Long-term effect of in vitro culture of mouse embryos with serum on mRNA expression of imprinting genes, development, and behavior.</a> Proc. Natl. Acad. Sci USA. 101, 5880-5885 (2004).</li><li>Bermejo-Alvarez, P., Roberts, R. M., Rosenfeld, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+glucose+concentration+during+in+vitro+culture+of+mouse+embryos+on+development+to+blastocyst,+success+of+embryo+transfer,+and+litter+sex+ratio.">Effect of glucose concentration during in vitro culture of mouse embryos on development to blastocyst, success of embryo transfer, and litter sex ratio.</a> 79, 329-336 (2012).</li><li>Tarkowski, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experiments+on+the+development+of+isolated+blastomers+of+mouse+eggs.">Experiments on the development of isolated blastomers of mouse eggs.</a> Nature. 184, 1286-1287 (1959).</li><li>Whittingham, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilization+of+mouse+eggs+in+vitro.">Fertilization of mouse eggs in vitro.</a> Nature. 220, 592-593 (1968).</li><li>McLaren, A., Biggers, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+development+and+birth+of+mice+cultivated+in+vitro+as+early+as+early+embryos.">Successful development and birth of mice cultivated in vitro as early as early embryos.</a> Nature. 182, 877-878 (1958).</li><li>McLaren, A., Michie, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+transfer+of+fertilized+mouse+eggs+to+uterine+foster-mothers.+I.+Factors+affecting+the+implantation+and+survival+of+native+and+transferred+eggs.">Studies on the transfer of fertilized mouse eggs to uterine foster-mothers. I. Factors affecting the implantation and survival of native and transferred eggs.</a> J. Exp. Biol. 33, 394-416 (1956).</li><li>Goto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fate+of+embryos+transferred+into+the+uterus.">The fate of embryos transferred into the uterus.</a> J. Assist. Reprod. Gen. 10, 197-201 (1993).</li><li>Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse Embryo: A Laboratory Manual. , (2003).</li><li>Chin, H. J., Wang, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utero-tubal+transfer+of+mouse+embryos.">Utero-tubal transfer of mouse embryos.</a> Genesis. 30, 77-81 (2001).</li><li>Ramirez, M. A., Fernandez-Gonzalez, R., Perez-Crespo, M., Pericuesta, E., Gutierrez-Adan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+stem+cell+activation,+culture+media+of+manipulated+embryos,+and+site+of+embryo+transfer+in+the+production+of+F0+embryonic+stem+cell+mice.">Effect of stem cell activation, culture media of manipulated embryos, and site of embryo transfer in the production of F0 embryonic stem cell mice.</a> Biol. Reprod. 80, 1216-1222 (2009).</li><li>Miller, A. M., Wright-Williams, S. L., Flecknell, P. A., Roughan, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+abdominal+and+scrotal+approach+methods+of+vasectomy+and+the+influence+of+analgesic+treatment+in+laboratory+mice.">A comparison of abdominal and scrotal approach methods of vasectomy and the influence of analgesic treatment in laboratory mice.</a> Lab. Anim. 46, 304-310 (2012).</li><li>Flecknell, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory Animal Anaesthesia. , (2009).</li><li>Erhardt, W., Hebestedt, A., Aschenbrenner, G., Pichotka, B., Blumel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+with+various+anesthetics+in+mice+(pentobarbitone+ketamine-xylazine,carfentanyl-etomidate).">A comparative study with various anesthetics in mice (pentobarbitone ketamine-xylazine,carfentanyl-etomidate).</a> Res. Exp. Med. 184, 159-169 (1984).</li><li>Tarin, D., Sturdee, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+anaesthesia+of+mice:+evaluation+of+tribromo-ethanol,+ether,+halothane+and+methoxyflurane+and+development+of+a+reliable+technique.">Surgical anaesthesia of mice: evaluation of tribromo-ethanol, ether, halothane and methoxyflurane and development of a reliable technique.</a> Lab. Anim. 6, 79-84 (1972).</li><li>Zeller, W., Meier, G., Burki, K., Panoussis, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adverse+effects+of+tribromoethanol+as+used+in+the+production+of+transgenic+mice.">Adverse effects of tribromoethanol as used in the production of transgenic mice.</a> Lab. Anim. 32, 407-413 (1998).</li><li>Lieggi, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+and+safety+of+stored+and+newly+prepared+tribromoethanol+in+ICR+mice.">Efficacy and safety of stored and newly prepared tribromoethanol in ICR mice.</a> Contemp. Top. Lab. Anim. Sci. 44, 17-22 (2005).</li><li>Lieggi, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+preparation+methods+and+storage+conditions+of+tribromoethanol.">An evaluation of preparation methods and storage conditions of tribromoethanol.</a> Contemp. Top. Lab. Anim. Sci. 44, 11-16 (2005).</li><li>Meyer, R. E., Fish, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+tribromoethanol+anesthesia+for+production+of+genetically+engineered+mice+and+rats.">A review of tribromoethanol anesthesia for production of genetically engineered mice and rats.</a> Lab. Anim. 34, 47-52 (2005).</li><li>Chatot, C. L., Lewis, J. L., Torres, I., Ziomek, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+1-cell+embryos+from+different+strains+of+mice+in+CZB+medium.">Development of 1-cell embryos from different strains of mice in CZB medium.</a> Biol. Reprod. 42, 432-440 (1990).</li><li>Quinn, P., Barros, C., Whittingham, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+hamster+oocytes+to+assay+the+fertilizing+capacity+of+human+spermatozoa.">Preservation of hamster oocytes to assay the fertilizing capacity of human spermatozoa.</a> J. Reprod. Fertil. 66, 161-168 (1982).</li><li>Dios Hourcade, d. e., Perez-Crespo, J., Serrano, M., Gutierrez-Adan, A., A, B., Pintado, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+development+of+mice+morulae+after+storage+in+non-frozen+conditions.">In vitro and in vivo development of mice morulae after storage in non-frozen conditions.</a> Reprod. Biol. Endocrinol. 10, 62 (2012).</li></ol>

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</...

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 border="0" cellpadding="0" cellspacing="0" width="1234">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="29">
			<td height="29" style="height:29px;width:232px;">Ketamine</td>
			<td style="width:111px;">VEDCO</td>
			<td style="width:203px;">Ketaved ANADA 200-257</td>
			<td style="width:691px;">To be ordered by a licensed veterinarian.</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:232px;">Xylazine</td>
			<td style="width:111px;">Lloyd laboratories</td>
			<td style="width:203px;">Anased NADA #139-236</td>
			<td style="width:691px;">To be ordered by a licensed veterinarian.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:232px;">Buprenorphine</td>
			<td style="width:111px;">Generic</td>
			<td style="width:203px;">NDC 400-42-010-01</td>
			<td style="width:691px;">To be ordered by a licensed veterinarian.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Eye ointment</td>
			<td style="width:111px;">Novartis</td>
			<td style="width:203px;">Genteal</td>
			<td style="width:691px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:232px;">Antibiotic</td>
			<td style="width:111px;">Pfizer</td>
			<td style="width:203px;">Clavamox NADA #55-101.</td>
			<td style="width:691px;">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 height="21">
			<td height="21" style="height:21px;width:232px;">Dressing serrated forceps</td>
			<td style="width:111px;">ROBOZ</td>
			<td style="width:203px;">RS-8120</td>
			<td style="width:691px;">Any medium size surgical-grade steel straight forceps will work.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Micro dissecting serrated forceps</td>
			<td style="width:111px;">ROBOZ</td>
			<td style="width:203px;">RS-5137</td>
			<td style="width:691px;">These ones are curved at a 90&#186; angle. Straight forceps can be used if preferred.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Slight curved micro dissection forceps</td>
			<td style="width:111px;">ROBOZ</td>
			<td style="width:203px;">RS-5136</td>
			<td style="width:691px;">This model is particularly useful to hold the oviduct.</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:232px;">Scissors</td>
			<td style="width:111px;">ROBOZ</td>
			<td style="width:203px;">RS-5880</td>
			<td style="width:691px;">Any regular surgical grade steel small straight scissors will work.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">27G needles</td>
			<td style="width:111px;">Beckton-Dickinson</td>
			<td style="width:203px;">305136</td>
			<td style="width:691px;">Smaller needles (30G) can be also used. 25G may be a bit too big.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:232px;">Clip applier</td>
			<td style="width:111px;">MiKRon</td>
			<td style="width:203px;">42763</td>
			<td style="width:691px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:232px;">9 mm Clips</td>
			<td style="width:111px;">MiKRon</td>
			<td style="width:203px;">427631</td>
			<td style="width:691px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Clip remover</td>
			<td style="width:111px;">MiKRon</td>
			<td style="width:203px;">7637</td>
			<td style="width:691px;">Two pairs of teeth forceps (ROBOZ RS-8160) can be used instead.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:232px;">Suture needle holder</td>
			<td style="width:111px;">ROBOZ</td>
			<td style="width:203px;">RS-7820</td>
			<td style="width:691px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:232px;">Suture</td>
			<td style="width:111px;">Dowist Gell</td>
			<td style="width:203px;">5-0 Dexon S 7204-21</td>
			<td style="width:691px;">Can be substituted for any 4-0 to 6-0 absorbable suture with a narrow curved needle.</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:232px;">Glass capillaries</td>
			<td style="width:111px;">VWR</td>
			<td style="width:203px;">100 ul calibrated pipettes 53432-921</td>
			<td style="width:691px;">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.</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:232px;">Burner</td>
			<td style="width:111px;">KISAG AG</td>
			<td style="width:203px;">Typ 2002</td>
			<td style="width:691px;">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 height="105">
			<td height="105" style="height:105px;width:232px;">Stereomicroscope</td>
			<td style="width:111px;">Leica</td>
			<td style="width:203px;">MZFLIII</td>
			<td style="width:691px;">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.&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:232px;">Fiber optics ilumination</td>
			<td style="width:111px;">Dolan Jenner</td>
			<td style="width:203px;">Fiber lite</td>
			<td style="width:691px;">To iluminate the surgical area. There are different systems available.</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:232px;">Warm stages</td>
			<td style="width:111px;">American scope</td>
			<td style="width:203px;"><a href="https://exch.mail.umd.edu/owa/redir.aspx?C=1qTJKejamUSfudeYG_MlZHvTY3dsJM9Iaqr2SnwGD4ECWGzB8e8Rpg--_L7u0vJmyatXQJzGQ0k.&amp;URL=http%3a%2f%2fstore.amscope.com%2ftcs-100.html" target="_blank">http://store.amscope.com/tcs-100.html</a></td>
			<td style="width:691px;">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 height="20">
			<td height="20" style="height:20px;width:232px;">Culture dishes for embryo manipulation</td>
			<td style="width:111px;">Falcon</td>
			<td style="width:203px;">353001</td>
			<td style="width:691px;">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...

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

Research

JoVE Journal

Biology

47.4K 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

Nuclear Transfer into Mouse Oocytes

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).

This movie and the protocol are intended to help learning nuclear transfer.

小鼠卵母细胞的细胞核移植到

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

科研

生物学

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

在鸡胚神经诱导的含量为

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

在电生理记录果蝇胚胎

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

研究有丝分裂的显微注射技术果蝇合胞体胚胎

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

在早期鸡胚跟踪形态组织变形

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

鼠胚体外发育无血清全胚胎培养系统

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

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 ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

小鼠模型的验证扰乱LINC配合物在细胞特异性的方式

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...