This research was supported in part by the University of Georgia, and a grant (HD071330) from the National Institutes of Health to MMV.

该协议被批准的机构动物护理和使用委员会（IACUC）在佐治亚大学。 1.准备<ol><li>对卵母细胞培养，购买或新鲜制备最低必需培养基（MEM）和补充用3毫克/毫升牛血清白蛋白（BSA），如表1中概述 。将聚苯乙烯瓶上的装载平衡（皮重至零）。加入所有试剂，除了BSA，为了带来与MQ水最终体积（重量）总共250克。然后加入BSA，让溶解，过滤消毒。 注意：所述的MEM培养基中，需要的平衡和细胞温育，用5％ 的 CO 2，5％O 2和90％的N 2的医疗用气体混合物。然而，其他的媒体（例如，CZB）可用于在允许在5％CO 2温育细胞的大气条件下。 </li><li>对卵母细胞显微注射，购买或准备M2网上平台。 </li><li>准备p统治马血清促性腺激素（PMSG）至5 IU / 100微升的浓度。 </li><li>购买和重构的siRNA股1μM的一个工作浓度。 </li></ol> 2.小​​鼠卵母细胞采集<ol><li> 第1天：为了刺激卵泡发育，提高排卵前卵泡的数量，辖5 IU PMSG的腹腔内雌性小鼠48小时之前，卵母细胞的采集。 </li><li> 第3天：卵母细胞的收集和培养，设立了35个100mm培养菜肴用3ml MEM / BSA的添加1微克/毫升米力农。这种磷酸二酯酶抑制剂维持卵母细胞在前期，我逮捕，并防止生发泡破裂（GVBD）。平衡的培养皿在医疗气体混合物至少15分钟。 需要在整个卵母细胞的采集，以及卵母细胞显微注射和24小时的文化持有期后的siRNA米力农补充媒体：注意。 </li><li>检索使用已建立实验室雌性小鼠卵巢实行15和转移到新的培养皿预热和平衡MEM / BSA /米力农。 </li><li>放置在一个立体的卵母细胞的收集阶段培养皿。发布丘卵母细胞复合体（COC的）通过手动穿刺卵泡与27G的针头。固定一个卵巢到培养皿的底部，与连接到一个1ml注射器一个27G的针头，并用第二针穿刺所有大卵泡。重复每个卵巢。 </li><li>使用标准的嘴移液过程或卵母细胞抽吸等方式，收集了由2层以上紧凑的卵丘细胞包围着所有的卵母细胞。转移COC的一个新盘，37℃下1小时。 </li><li>小心取出周围的卵丘细胞反复轻柔吹打用1毫升吸管设置为750微升的吸液容量。重复移液步约12倍并允许卵母细胞恢复5分钟。继续以这种方式，直到所有的或大部分的卵丘细胞已被删除。传送裸卵到另一培养皿中，并允许平衡在37℃下进行15分钟。 </li><li>分配裸露的卵母细胞分成三个实验组，以用于：特异性siRNA（ⅰ）显微注射针对感兴趣的靶，（ⅱ）注射的非特异性（对照）的siRNA，和（iii）一个未注射对照组占培养条件。 </li></ol> 3.卵母细胞显微注射<ol><li>设置培养皿补充有米力农（1微克/毫升）3毫升M2媒体为卵母细胞注射。制备含MEM / BSA /米力农清洗并注入卵母细胞的后续培养培养皿。 注意：M2培养基包含HEPES缓冲液，并且需要预先升温在37℃大气条件下15分钟。 MEM需要预热和平衡使用医用气体作为描述d以上。 </li><li>准备显微注射系统。加载注射针，用5微升1μM的特异siRNA溶液。固定固定吸管和注射针显微注射系统的显微操作，并设置了校准的体积和压力的注射装置。 </li><li>放置一个200μl的微滴的M2培养基上3厘米培养皿的盖的内侧，并添加一个卵母细胞用于设置和显微注射系统的对齐。 </li><li>利用卵母细胞为指导，调整持有和注射针头的位置。施加负压到保持吸管轻轻固定卵母细胞和调节注射针的位置的小区的最宽直径。调整设置，使大约10 PL的siRNA溶液的注射任何地方进入卵母细胞的细胞质中。 <img alt="图1" src="/files/ftp_upload/53586/53586fig1.jpg" /> 图1.卵母细胞显微注射成立。（ <stro纳克&gt;200μl的微滴的M2媒体的放置在盖子从一个3厘米的培养皿，用固定吸管和注射针定位在左侧和右侧，分别A）的代表性的图像。 （二）集团前期，我被捕前显微注射的卵母细胞（GV期）。 <a href="https://www.jove.com/files/ftp_upload/53586/53586fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </li><li>返回到盖体视显微镜阶段，并添加200μl的微滴新鲜M2的介质。添加组卵母细胞（〜10），以微滴通过口移液，或其他方法，然后返回该盖与微滴的显微注射阶段。 <ol><li>继续microinject个人卵母细胞。固定一根卵母细胞与保持吸管，缓缓插入注射针的尖端进入细胞质和驱逐的siRNA溶液的注射体积。仔细缩回注射针和莫已经注入的卵母细胞的位置在微滴的底部。动失败的卵母细胞注射到一位置，在微滴的顶部。 </li><li>每个卵母细胞重复注射过程。以保持最佳的卵母细胞的生存能力，microinject只有少数的卵母细胞在一个时间。确保这一过程不超过几分钟采取更多，以防止温度和pH的变化在微滴。 </li></ol></li><li>抽吸成功注入的卵母细胞或小心地移动所述盖与微滴喷射的卵母细胞，以立体显微镜。传输所注入的卵母细胞存活由口移液或替代方法，为MEM / BSA /米力农介质洗并平衡在37℃。 </li><li>使用另一组卵母细胞，用新的注射针装入为对照组的非特异性的siRNA重复显微注射过程。洗MEM / BSA /米力农和转让的卵母细胞，以一个单独的培养皿。 </li><li>文化各组医疗气体气氛下的卵母细胞24小时的MEM / BSA /米力农，在37℃。 注：需要高效的靶mRNA /蛋白枯竭的“米力农块”文化时期。 </li></ol> 4，卵母细胞培养的卵母细胞减数分裂<ol><li> 第4天：对于每个实验组的卵母细胞建立4培养皿（35毫米）用3ml MEM / BSA中。补充每组一个媒体盘，用10％（高品质）胎牛血清（FBS）的用于卵母细胞成熟。允许所有培养皿在37℃，平衡医疗气体混合物。 </li><li>从&#39;米力农块“释放的卵母细胞，依次在含有MEM / BSA的碗碟洗涤卵母细胞3次，然后将卵母细胞转移到含有10％FBS的培养基中成熟的菜。培养卵母细胞的所有组17小时，在37℃，以使恢复和减数分裂的进展。 </li><li> 第5天：卵母细胞的FIXa准备解决方案灰，主要包括：（ⅰ）4％低聚甲醛（PFA）的PEM缓冲液（100毫摩尔PIPES [pH值6.9]，1mM的MgCl 2的，1mM的EGTA）用0.5％Triton-X 100，和（ii）的PBS补充有5 ％FBS中，将用于洗涤和阻断的卵母细胞。 注意：这些解决方案需要预先温热至37℃之前，卵母细胞的固定。 </li><li>对卵母细胞的固定（在17小时培养后），迅速通过含有750微升口移液或替代方法为单独的孔中（一个4孔培养皿的）转移的每个实验组的卵母细胞的预热4％PFA溶液和在37 °下进行1小时。在750微升预热的PBS含有5％FBS随后清洗各组卵母细胞3次（15分钟的每个）。 </li><li>阻止卵母细胞在200微升PBS / 5％FBS O / N在4°C到非特异性抗体减少结合。 </li></ol> 5.免疫荧光分析<ol><li> 第6天： 注意：免疫染色是在一个多井板和OOC完成ytes被串行传送到顺序井，含有各自的抗体和洗涤溶液。 48或96孔板可以使用，简单地基于以及大小调整溶液体积。对于96孔板用每孔200μl的。卵母细胞被转移到不同的解决方案中的顺序如下：初级抗体溶液 - 3洗井 - 二级抗体溶液 - 3洗涤井。为了减少曝光的板可以用铝箔纸覆盖。 制备新鲜的PBS / 5％FBS和使用此解决方案，制备抗体稀释液（ 例如，兔抗粒周蛋白（1 / 1,000），小鼠抗微管蛋白（1 / 1,000））。 </li><li>转移的每个实验组与初级抗体溶液含有200微升（或100微升，如果少体积期望）单独孔的固定卵母细胞并覆盖井用封口膜。根据最佳抗体的特定条件下孵育（例如，37℃1小时，或4℃的CO / N）。 注意：一抗dilutioN作为以及具体的培养时间和温度需要测试，以确定所使用的不同抗体的最佳条件。 </li><li>温育与第一抗体后，清洗的卵母细胞三次在PBS / 5％FBS（每10-15分钟）。 </li><li>转移卵母细胞成含有荧光标记的二抗的溶液（例如，1/1000稀释的抗兔和抗小鼠抗体偶联有不同波长的荧光染料）。孵育1小时，在37℃。 </li><li> （ - 15每分钟10）在PBS / 5％FBS的洗涤卵母细胞三次。 </li><li>在最后的洗涤步骤后，转移到卵母细胞干净的载玻片上，轻轻地吸出任何过量的洗涤溶液。这将固定在玻璃表面上的卵母细胞。添加8微升安装介质（含DAPI），小心地覆盖安装媒体22毫米×22毫米遮滑。降低盖玻片缓慢，以避免俘获气泡和/或损坏的卵母细胞。 注意：可替换地，保持卵母细胞进行共聚焦显微镜分析的3-维属性，添加少量的100微米的玻璃珠（与凡士林混合）到盖玻片的角部安装在滑动之前。 </li><li>商店载玻片在4℃，或者进行到减数分裂进展的评估，以及使用配备了必要的过滤器在荧光显微镜来匹配所用的第二抗体的表达水平和亚细胞蛋白质定位进行分析。 </li></ol>

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The authors have nothing to disclose

尽管有多种方法外源核酸转移进入体细胞，例如电穿孔和转染，显微注射是用于递送RNA分子的转录成静止小鼠卵母细胞的最佳方法。目前的协议提供了一种有效的方法用于体外耗尽特异mRNAs，使不同的主轴和/或微管组织中心相关因子中的卵母细胞的功能测试。这种做法会导致效率的成绩单枯竭和高度的适应性。虽然的siRNA被用于特异性靶向PCNT转录，类似的条件，可以采用靶向感兴趣?...

减数分裂是发生在配子（卵子和精子），涉及两个连续的部门不干预DNA合成减数分裂我和减数分裂-II， 分别为1时分离的同源染色体和姊妹染色单体独特的分裂过程。在卵母细胞在减数分裂中染色体分离错误可能导致非整倍体，它是由胚胎在受精过程中继承。值得注意的是，非整倍体在发育中的胚胎的发生率的增加而前进产妇年龄和是先天性出生缺陷的主要原因，以及在妇女妊娠1,2-损失，因此，强调理解非整倍体的过程中减数分裂的分子基础的重要需求。 在细胞分裂，染色体分离是关键取决于微管主轴装置，并建立稳定的染色体微管相互作用进行正确连接到对面纺锤体组装极。重要的是，减数分裂纺锤体形成在哺乳动物卵母细胞不同于有丝分裂中的体细胞，并通过独特的微管组织中心（MTOCs）缺乏中心粒3,4-调节。必要的微核和组织必需的蛋白质定位于卵母细胞MTOCs，包括γ - 微管蛋白的催化微管组装。另外，粒周功能作为必要支架蛋白，其结合和锚γ微管蛋白以及其他因素在MTOCs 5。值得注意的是，我们的研究表明，主要的微管组织中心相关蛋白的耗尽扰乱减数分裂纺锤体组织，并导致在卵母细胞的染色体分离的错误，这是不充分的主轴组件的检查点（SAC）6,7-解决。因此，在主轴的稳定性的缺陷，即不会触发减数分裂阻滞，姿势在促进非整倍体一个显著风险。尽管在纺锤体组装和组织下，卵母细胞微管组织中心PROT的重要作用EIN组成和功能仍然知之甚少。 测试特定的目标蛋白质的功能在哺乳动物卵母细胞是具有挑战性的，因为细胞变得转录静止恢复减数分裂8,9前不久。因此，排卵前的卵母细胞依靠产妇基因店恢复减数分裂和支持减数分裂和受精10,11后的第一次卵裂的分裂。 RNA干扰（RNAi）介导的mRNA转录物在哺乳动物卵母细胞退化的功效是公认的，并在减数分裂成熟招募翻译母系RNA是特别适合于siRNA的靶向12-14中 。因此，短干扰RNA显微注射（的siRNA）为卵母细胞提供了一个有价值的方法来耗尽目标基因进行功能测试。 在这里，我们描述了小鼠卵母细胞和具体的transcri siRNA-介导的耗尽的分离方法点测试的必要微管组织中心相关蛋白的功能，粒周。此外，我们描述免疫荧光分析条件来评估减数分裂纺锤体形成在卵母细胞。

<table border="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pregnant Mare&#39;s Serum Gonadotropin (PMSG)</td>
			<td>EMD Biosciences</td>
			<td>367222</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimal Essential Medium (MEM)</td>
			<td></td>
			<td></td>
			<td>*Recipe outlined in Table 1</td>
		</tr>
		<tr>
			<td>Earle&#39;s Balanced Salt Solution (10x)</td>
			<td>Sigma</td>
			<td>E-7510</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>S-5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvic Acid, sodium salt&#160;</td>
			<td>Sigma</td>
			<td>P-5280</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G, potassium salt&#160;</td>
			<td>Sigma</td>
			<td>P-7794</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin Sulfate&#160;</td>
			<td>Sigma</td>
			<td>S-9137</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine&#160;</td>
			<td>Sigma</td>
			<td>G-8540</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, disodium salt dihydrate&#160;</td>
			<td>Sigma</td>
			<td>E-4884</td>
			<td></td>
		</tr>
		<tr>
			<td>Essential Amino Acids (50x)</td>
			<td>Gibco&#160;</td>
			<td>11130-051</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Vitamin Mixture (100x)</td>
			<td>Sigma</td>
			<td>M-6895</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red solution</td>
			<td>Sigma</td>
			<td>P-0290</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A1470</td>
			<td></td>
		</tr>
		<tr>
			<td>Milrinone</td>
			<td>Sigma</td>
			<td>M4659</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30070.01</td>
			<td></td>
		</tr>
		<tr>
			<td>EmbryoMax M2 Media with Hepes</td>
			<td>EMD Millipore</td>
			<td>MR-015-D</td>
			<td></td>
		</tr>
		<tr>
			<td>siRNAs targeting Pericentrin</td>
			<td>Qiagen</td>
			<td>GS18541</td>
			<td></td>
		</tr>
		<tr>
			<td>Negative control siRNAs&#160;</td>
			<td>Qiagen</td>
			<td>SI03650318</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (16% solution)</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Sigma</td>
			<td>T-8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Hyclone</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Pericentrin (rabbit)</td>
			<td>Covance</td>
			<td>PRB-432C</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-acetylated a-tubulin (mouse)</td>
			<td>Sigma</td>
			<td>T-6793</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbbit Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-21430</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti -mouse Alexa Fluor 555</td>
			<td>Invitrogen</td>
			<td>A-11017</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Major Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope (SMZ 800)</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Upright Fluorescent Microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Nikon&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Femtojet Micro-injections System</td>
			<td>Eppenforf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro manipulators</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-injection needles (femtotips)</td>
			<td>Eppendorf</td>
			<td>930000035</td>
			<td></td>
		</tr>
		<tr>
			<td>Holding pipettes (VacuTip)</td>
			<td>Eppendorf</td>
			<td>930001015</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasticware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35mm culture dishes</td>
			<td>Corning Life Sciences</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr>
			<td>4-well plates</td>
			<td>Thermo Scientific</td>
			<td>176740</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plates</td>
			<td>Corning Life Sciences</td>
			<td>3367</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 mm CA Filter System</td>
			<td>Corning Life Sciences</td>
			<td>430768</td>
			<td></td>
		</tr>
	</tbody>
</table>

meiotic spindle assessment in mouse oocytes by sirna-mediated silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

siRNA的显微注射提供了一种有效的方法对mRNA降解和随后的蛋白耗竭的卵母细胞，从而使不同的目标因子在体外的高效率和高特异性的功能测试。随后，免疫荧光被用于特定的表型分析以及验证蛋白耗竭的siRNA注射卵母细胞。在当前的例子，用DAPI个别的卵母细胞的抗微管蛋白和抗粒周蛋白抗体荧光标记一起启用：粒周枯竭（i）认可，以及（ii）以控制这两个染色质和减数分裂纺锤体结构进行...

Watch this Scientific Journal Video about Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing at JoVE.com

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

在这里，我们提出了一个协议，用于特定的siRNA介导的mRNA枯竭其次是免疫荧光分析来评估减数分裂纺锤体组装和组织在小鼠卵母细胞。这个协议是适合体外转录耗尽的和不同的主轴和/或微管组织中心相关因子中的卵母细胞的功能评价。

减数分裂纺锤体评估在小鼠卵母细胞的siRNA介导沉默

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

meiotic-spindle-assessment-mouse-oocytes-sirna-mediated

Research

JoVE Journal

Biology

11.6K 次观看.  College of Veterinary Medicine, University of Georgia. 在这里，我们提出了一个协议，用于特定的siRNA介导的mRNA枯竭其次是免疫荧光分析来评估减数分裂纺锤体组装和组织在小鼠卵母细胞。这个协议是适合体外转录耗尽的和不同的主轴和/或微管组织中心相关因子中的卵母细胞的功能评价。 

视频: Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

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

Retrieval of Mouse Oocytes

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus Genetic Stock Center (http://stkctr.biol.sc.edu), we are characterizing the salient differences needed to develop this system.  A primary goal has been to optimize oocyte/early embryo retrieval.

This protocol illustrates the technique for extracting oocytes or early-stage fertilized embryos from the oviduct of mice. The ability to identify the infindibulum and insert a blunt end needle into it is essential to correctly performing the procedure.

检索小鼠卵母细胞

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus ...

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse embryos could be frozen and stored at -196 &deg;C and, a few years later, Parkening et al. 3 reported the birth of live offspring resulting from in vitro fertilization (IVF) of cryopreserved oocytes. Since then, the use of cryopreservation techniques has rapidly spread to become an essential component in the practice of human and animal assisted reproduction and in the conservation of animal genetic resources. Currently, there are two main methods used to cryopreserve oocytes and embryos: slow freezing and vitrification. A wide variety of approaches have been used to try to improve both techniques and millions of animals and thousands of children have been born from cryopreserved embryos. However, important shortcomings associated to cryopreservation still have to be overcome, since ice-crystal formation, solution effects and osmotic shock seem to cause several cryoinjuries in post-thawed oocytes and embryos. Slow freezing with programmable freezers has the advantage of using low concentrations of cryoprotectants, which are usually associated with chemical toxicity and osmotic shock, but their ability to avoid ice-crystal formation at low concentrations is limited. Slow freezing also induces supercooling effects that must be avoided using manual or automatic seeding 4. In the vitrification process, high concentrations of cryoprotectants inhibit the formation of ice-crystals and lead to the formation of a glasslike vitrified state in which water is solidified, but not expanded. However, due to the toxicity of cyroprotectants at the concentrations used, oocytes/embryos can only be exposed to the cryoprotectant solution for a very short period of time and in a minimum volume solution, before submerging the samples directly in liquid nitrogen 5. In the last decade, vitrification has become more popular because it is a very quick method in which no expensive equipment (programmable freezer) is required. However, slow freezing continues to be the most widely used method for oocyte/embryo cryopreservation. In this video-article we show, step-by-step, how to collect and slowly freeze hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

In this video-article we present a step-by-step demonstration on how to collect and cryopreserve hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

收集和仓鼠卵母细胞和小鼠胚胎冷冻保存

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse ...

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 ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

流动小鼠减数分裂细胞的流式细胞仪净化

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

活细胞钙成像相结合的siRNA介导的基因沉默标识CA 2 +泄漏通道内质网膜和监管机制

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

农杆菌介导的病毒诱导的基因沉默含量在棉花

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

长期沉默的1S Intersectin的在小鼠肺的特异性siRNA，通过阳离子脂质体重复交货。通过电子显微镜的击倒效果评价

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...