这项工作是在理查德M.舒尔茨的实验​​室进行。作者还要承认迈克尔兰普森，构思着丝粒计数他共聚焦显微镜检测和访问。邓丽君蒋介石和弗朗西斯邓肯协助优化的着丝粒计数检测。保斯坦支持HD022681（有效值）和卡伦辛德勒是HD055822支持。

1。小鼠卵母细胞的收集<ol><li>为了最大限度地从每个鼠标中分离的卵泡数，腹腔注入性成熟的雌性小鼠（我们使用6周龄CF - 1小鼠从哈伦）5 IU孕马血清促性腺激素（PMSG）。 </li><li>准备收集培养基（MEM / PVP）（3毫升/鼠标），增加至2.5微米的米力农和温暖，在37 ° C。米力农是一个磷酸二酯酶抑制剂，保持减数分裂逮捕一次卵母细胞从卵泡中删除。另外，3 - 异丁基-1 - 甲基黄嘌呤（IBMX）（0.2毫米）或丁酰环磷酸腺苷（dbcAMP）（100微克/毫升），可以使用。准备谷氨酰胺（1毫米）和米力农（2.5微米），加入1毫升的CZB培养液中，并允许它至少一个小时，在孵化器的平衡。在培养皿中与矿物油覆盖，设置每个microdrops。 CZB盘放入孵化器，而MEM / PVP的菜仍然幻灯片温暖之外。其他收集和培养液，均为市售（M2和M16从专业媒体或Sigma）也经常使用。 </li><li> PMSG吸约48 h后，牺牲了鼠标使用的CO 2镇静颈椎脱位，解剖卵巢和它们放入一个含有预热MEM / PVP +米力农（MEM / PVP + M）的表面玻璃。 </li><li>使用胰岛素注射器1毫升，锚卵巢菜和27号（或缝）是固定在一起的针头刺破几次释放卵泡。 </li><li>通过解剖镜下的同时，收集卵丘卵母细胞复合体，使用口操作的玻璃吸管。这是有益的，有大量的对比显微镜。另外，组织和细胞的培养基中含有可吸管塑料培养皿的盖子。只收集大，卵泡，而不是较小的前卵泡或裸露的卵母细胞。一旦收集，转移配合物的MEM / PVP + M microdrop幻灯片温暖和石油。 </li><li>用一个小吸管（略高于卵母细胞的直径较大），移液器向上和向下的复合物分离的卵丘细胞。裸露的卵母细胞与成一个CZB +米力农（CZB + M）microdrop，较大的移液器和孵化器进行传输。 </li><li>允许卵母细胞恢复前至少1 h在孵化器操纵。 </li></ol> 2。卵母细胞显微注射<ol><li>机械拉马拉硼硅玻璃毛细管注射移液器。我们使用以下设置一个火焰 - 布朗微量拉马（模型P - 97）：P = 500，热= 300，拉= 150，VEL = 100，时间= 150。 </li><li>准备1井室幻灯片上放置尽可能接近的MEM / PVP + M 5μL下降0.5μL注射的首选材料下降（例如siRNA的，cRNA的，吗啉代寡核苷酸等）的微量注射平台有媒体室删除。盖与矿物油和显微镜舞台上（图1）。 </li><li>到持有人的地位下降到MEM / PVP + M广场注入和控股移液器打开连接到picoinjector和抗振动表上的氮罐。打开轻轻拍打，对控股吸管却使中等注射针的尖端。如果开口过大，中型移动并迅速和中针会杀死细胞。太小开幕移液器往往容易堵塞。 </li><li>转移卵母细胞（5-10）从孵化器平台的MEM / PVP + M在下降。注射前，设置picoinjector。我们用我们picoinjector（PLI - 100型从哈佛器械）以下参数：PBal = 2.5-3.5 PSI，PInj = 7.8磅，PClear = 10-12 PSI，时间= 3秒。 5-10 PL注射量的结果。 </li><li>虽然按CLEAR键，将注射针头的物质下降，并填写。它返回到中期下降。 </li><li>使用拿着吸管捕捉到的卵母细胞和的注射针，卵母细胞和控股吸管沿X轴对齐。 </li><li>更高的放大倍率下，提前通过小心，以避免核的卵母细胞的注射针。质膜一旦被刺穿，按注入。注射后，退出针。释放卵母细胞和重复。一旦所有的卵母细胞注入，返回他们的孵化器。保持在CZB + M所需的时间，这个时间是取决于实验目的（即siRNA和吗啉击倒（最多24个小时），过度表达（3-12小时））。请注意，超过24小时的孵化，将危及母细胞成熟。 </li></ol> 3。卵母细胞成熟<ol><li>孵育后，通过CZB数滴（成熟介质）洗卵母细胞，并在孵化器的石油和地点转移到了成熟的中型microdrop。 </li><li>允许16 h完全成熟吨<html> Ø第二减数分裂中期。 </li></ol> 4。倍体分析<ol><li>准备CZB + monastrol（100微米），以及器官的培养皿中，水在外圈到750μL。 </li><li>通过数滴的CZB + monastrol洗净的鸡蛋，他们转移到机关培养皿。在孵化器中放置1小时</li><li>修正转移到一个含有2％多聚甲醛的PBS，在室温下20分钟的表面玻璃的鸡蛋。转移到另一个表面玻璃封闭液。这个菜可存放在4℃直至处理。 </li><li>转移到一个在室温下15分钟的表面玻璃通透的解决方案（PBS + 0.3％BSA + 0.1％TritonX - 100 + 0.02％，为NaN 3） 。冲洗通过大量封闭液（PBS + 0.3％BSA + 0.01吐温20 + 0.02％NaN的3）。 </li><li>该过程的其余部分是在96孔板在室温避光的湿盒的盖子上。 （〜25μL）滴放在圆形凹痕。将鸡蛋阻断15分钟的解决方案。 </li><li>标注着丝粒，转让鸡蛋下降阻塞1:40 CREST抗血清的解决方案。孵育至少1小时。通过封闭液3滴，在每个孵化15分钟洗净。 </li><li>鸡蛋传输阻塞解决方案包含Cy5的或Alexa的氟594 -共轭抗人IgG（1:200）孵育1h后下降。重复上述洗涤步骤，除了最后一步，包括Sytox绿色（1:5,000）在封闭液来检测染色体。安装在5μLVectashield。为防止破碎的鸡蛋，把凡士林在盖玻片将角落的小斑点。广场上的顶部和指甲油密封盖玻片。商店在幻灯片中于4℃，直到通过共聚焦显微镜进行处理的彗星。 </li><li>虽然与100X目标成像，捕捉在Z平面和形象的中期主轴整个地区的0.4微米的步骤。计数使用成像软件，如图像J（NIH）的着丝粒。 </li></ol> 5。代表性的成果： 图2是从整倍体卵子的Z轴投影。信息产业部中期，整倍体鼠标鸡蛋中含有20对染色体，因此有40个着丝粒。偶尔染色体不蔓延，尽管monastrol治疗。这种情况使得难以可靠地算着丝点，因此，我们不包括在我们的分析，这些鸡蛋。有时，它可能是一项挑战，以确定是否一个波峰免疫反应的“点”是1或2的着丝粒。使用程序（如图像J）是有用的，因为我们可以分析每个Z的部分，而仔细地注意到染色体的方向，有部分检测到其中一个“点”是“点”的像素强度。根据减数分裂纺锤体极体的相对位置，DNA的区域可以重叠，不应该包括在分析这些样本。 Unmanipulated，非整倍体的低利率（约1-2％），从繁殖幼鼠体内排卵的鸡蛋。然而，原因不理解，显微注射法和体外成熟过程中可以增加10％这样的速度向上。因此，关键是控制注入卵母细胞中包含任何显微注射研究。 <img src="/files/ftp_upload/2851/2851fig1.jpg" alt="图1"/> 图1。显微注射菜成立 。与上述注射液0.5μL滴5μL滴MEM / PVP + M室幻灯片。封面与矿物油。在这个例子中，有3滴为3种不同的注射解决方案的幻灯片是坐在显微镜阶段。左边是显微注射针和正确的，是拿着吸管。需要注意的是反映了针持有人。 <img src="/files/ftp_upload/2851/2851fig2.jpg" alt="图2"/> 图2。倍性的结果 。 Z -投影的一个整倍体的中期二蛋。 DNA的颜色在绿色和红色动粒。箭头指向2个不同的指示，动粒（＃17和＃18）的重叠染色单体武器。整倍体的鼠标鸡蛋中含有20双着丝粒（共40“点”）。非整倍体卵子将包含任何对这个数字的变化。如果这个过程进行中期MI卵母细胞，将有40双着丝粒（共80“点”）。 <img src="/files/ftp_upload/2851/2851table1.jpg" alt="表1"/> 表1。收集和显微注射液配方（MEM / PVP + M）。所有材料都胚胎文化品位和Sigma - Aldrich公司。过滤消毒通过0.22μm的聚偏氟乙烯过滤器（使用Milipore Stericups）和存储在4 ° C。 iles/ftp_upload/2851/2851table2.jpg“ALT =”表2“/&gt; 表2。 CZB培养基配方 。所有材料都胚胎文化品位和Sigma - Aldrich公司。过滤消毒通过0.22μm的聚偏氟乙烯过滤器（使用Milipore Stericups）和存储在4 ° C。

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微量注射的卵母细胞是一种强大的的方法来研究机制，规范减数分裂成熟10,11，12， 13。这种方法提供了一种经济的方式来测试假设一个投资大，在发展转基因和有针对性的的小鼠模型前。的卵母细胞的收集和显微注射技术，需要有更多的时间来掌握比典型的细胞生物学程序。与收集的具体障碍通常包括控制口吸管，拉动收集适当大小的玻璃吸管和体细胞的剥离和提高收集速度，以最少的时间，其中的卵母细胞孵化器以外的。我们建议做实验前练习多次。 microdrops之间传输，同时保持相同的细胞数量的卵母细胞，是一个伟大的方式，用这种方法成为舒适。 细胞死亡通常发生在学习显微注射。发生这种情况，有许多原因，包括注射过大的物质的量（即注射针头开放太大）击中细胞核注射针头，刺入对面的卵母细胞或物质注入卵母细胞是有毒的。注入卵母细胞缓冲练习，直到你的存活率是至少有50％是掌握这项技术的关键。卵母细胞如果没有成熟，这是可能的，米力农是不足够的稀释。我们建议通过许多大滴的米力农无CZB冲洗前的卵母细胞成熟。 倍体分析以下显微注射法是许多检测评估减数分裂成熟。我们在实验室中使用的其它常规分析，包括监测动力学卵母细胞通过减数分裂的进展，免疫荧光分析纺锤体的形成和染色体排列与卵子激活，或在体外受精，以评估的卵母细胞操纵14,15的发育后果，16， 17。

表的特定试剂： <table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>米力农</td><td> Sigma - Aldrich公司</td><td> M4659 </td><td>在2.5mm的悬浮在DMSO </td></tr><tr><td>矿物油</td><td> Sigma - Aldrich公司</td><td> M5310 </td><td>只有使用胚胎测试，无菌过滤</td></tr><tr><td> CREST自体血清</td><td> Immunovision </td><td> HCT - 0100 </td><td></td></tr><tr><td> Sytox绿</td><td> Invitrogen公司</td><td> 57020 </td><td></td></tr><tr><td>抗人Alexa的594 </td><td> Invitrogen公司</td><td> A - 11014 </td><td></td></tr><tr><td> Vectashield </td><td>媒介实验室</td><td> H - 100 </td><td></td></tr><tr><td>多聚甲醛</td><td> Polysciences </td><td> 577773 </td><td></td></tr><tr><td>从牛血清白蛋白</td><td> Sigma - Aldrich公司</td><td> A3294 </td><td></td></tr><tr><td> PMSG </td><td> Calbiochem </td><td> 367222 </td><td></td></tr><tr><td> Monastrol </td><td> Sigma - Aldrich公司</td><td> M8515 </td><td>在DMSO悬浮在100MM </td></tr><tr><td>吐温20 </td><td> Sigma - Aldrich公司</td><td> 274348 </td><td></td></tr><tr><td> TritonX - 100 </td><td> Sigma - Aldrich公司</td><td> X - 100 </td><td></td></tr></tbody></table>具体设备表： <table border="1"><tbody><tr><th>设备名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>喉舌</td><td> Biodiseno </td><td> MP - 001 - Y </td><td></td></tr><tr><td>表面玻璃</td><td>电子显微镜科学</td><td> 70543-30 </td><td></td></tr><tr><td>注射器</td><td>屋宇署</td><td> 309623 </td><td> 27G液1ml，1 / 2 </td></tr><tr><td> 60毫米培养皿</td><td>鹘</td><td> 351007 </td><td></td></tr><tr><td>玻璃巴斯德吸管</td><td> Fisher Scientific则</td><td> 13-678-200 </td><td></td></tr><tr><td>侧回暖</td><td> Fisher Scientific则</td><td></td><td>任何标准模型</td></tr><tr><td>解剖镜下</td><td></td><td></td><td>任何标准模型</td></tr><tr><td>商会幻灯片</td><td> NUNC </td><td> 177372 </td><td></td></tr><tr><td>毛细管</td><td>德拉蒙德</td><td> 1-000-0500 </td><td>微型</td></tr><tr><td>移液器拖轮</td><td>火焰山 - 布朗微量拉马</td><td></td><td> P - 97型</td></tr><tr><td>倒置显微镜</td><td>尼康</td><td></td><td>任何标准模型</td></tr><tr><td> Micromanipulators </td><td> Eppendorf公司</td><td></td><td>任何标准模型</td></tr><tr><td> Picoinjector </td><td>哈佛器械</td><td> PLI - 100型</td><td>任何标准模型</td></tr><tr><td> CO 2坦克</td><td></td><td></td><td>对于孵化器</td></tr><tr><td>氮气罐</td><td></td><td></td><td>对于表和喷油器</td></tr><tr><td>抗振动表</td><td>技术制造</td><td></td><td>任何标准模型</td></tr><tr><td>孵化器</td><td></td><td></td><td>任何标准模型</td></tr><tr><td>控股移液器</td><td> Eppendorf公司</td><td> 930001015 </td><td> Vacutip </td></tr><tr><td>共聚焦显微镜</td><td>徕卡</td><td></td><td>任何标准模型</td></tr><tr><td>解剖工具</td><td>精细科学的工具</td><td></td><td>任何标准模型</td></tr><tr><td>湿盒</td><td></td><td></td><td>我们使用特百惠</td></tr><tr><td>盖96孔板</td><td> NUNC </td><td> 263339 </td><td></td></tr><tr><td>显微镜载玻片</td><td> Fisher Scientific则</td><td> 12-544-3 </td><td></td></tr><tr><td>盖玻片</td><td>托马斯科学</td><td> 6663 - F10 </td><td>特别是显微镜的厚度会有所不同</td></tr><tr><td>中心以及器官培养皿</td><td> Fisher Scientific则</td><td> 353037 </td><td> 60 × 15mm的</td></tr></tbody></table>

mouse oocyte microinjection, maturation and ploidy assessment

染色体分离的失误，导致非整倍体细胞。非整倍体细胞，是与癌症相关的，但在配子，非整倍性导致不孕，流产或发育障碍，唐氏综合症。单倍体配子形式，通过特定物种，再加以减数分裂的发展计划。第一次减数分裂（MI）的独特的减数分裂，同源染色体分开，因为姐妹染色单体时保持连接。 reductional分工不能完全理解的原因，这是容易出错，是更常见的非整倍体在减数分裂II（MII）的错误或比男性减数分裂 1,2中的错误来源。 在哺乳动物的卵母细胞在心肌梗死前期与一个大型的，完整的生发泡（GV;核），才恢复减数分裂，当他们收到排卵线索逮捕。一旦减数分裂恢复，卵母细胞完成MI和接受不对称的细胞分裂，在信息产业部中期再次逮捕。鸡蛋将无法完成信息产业部，直到它们被精子受精。也可以接受卵母细胞减数分裂成熟建立在体外培养条件3。由于转基因及基因靶向小鼠突变体的产生是昂贵的，可能需要很长一段时间， 雌配子在体外的操作是一种更为经济和节省时间的的策略。 在这里，我们描述了隔离小鼠和前期被捕的卵母细胞为显微注射的方法。任何材料的选择可能引入的卵母细胞，但因为meiotically主管卵母细胞转录沉默4,5 cRNA的，而不是DNA，必须注射异位表达研究。为了评估套数，我们描述了在信息产业部卵的卵母细胞体外成熟的条件。从历史上看，染色体传播技术用于计数染色体数目6。这种方法在技术上是具有挑战性的，唯一确定hyperploidies的是有限的。在这里，我们描述了一个方法来确定低hyperploidies使用完整的鸡蛋7-8。此方法使用monastrol，一个动蛋白- 5抑制剂，折叠成一个单极主轴9从而分离染色体等个别动粒可以很容易地使用反波峰自身免疫性血清检测和计数的两极主轴。由于这种方法是在执行完整的鸡蛋，染色体不因操作失误而丢失。

Watch this Scientific Journal Video about Mouse Oocyte Microinjection, Maturation and Ploidy Assessment at JoVE.com

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

卵母细胞很容易出现非整倍体在减数分裂成熟，由于染色体分离中的错误。非整倍体的鸡蛋可能会导致不孕，流产或发育障碍唐氏综合症。在这里，我们描述的方法引入到卵母细胞和方法来研究细胞成熟和评估套数的首选材料。

小鼠卵母细胞显微注射，成熟和倍性评估

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

mouse-oocyte-microinjection-maturation-and-ploidy-assessment

Research

JoVE Journal

Biology

29.7K 次观看.  University of Pennsylvania. 卵母细胞很容易出现非整倍体在减数分裂成熟，由于染色体分离中的错误。非整倍体的鸡蛋可能会导致不孕，流产或发育障碍唐氏综合症。在这里，我们描述的方法引入到卵母细胞和方法来研究细胞成熟和评估套数的首选材料。

视频: Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever.  The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

埃及斑蚊胚胎显微注射获得转基因蚊子

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue ...

科研

生物学

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

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 ...

Single Oocyte Bisulfite Mutagenesis

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription1. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes2. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting.
One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level3. Development of the bisulfite mutagenesis assay has advanced from those originally reported4-6 towards ones that are more sensitive and reproducible7. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment8. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos9. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos10. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos.
Taking clues from agarose embedding of minute DNA amounts including oocytes11, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis12. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos.

Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.

单卵母细胞的亚硫酸氢钠诱变

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for ...

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.
The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).
For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular&#160;cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red&#160;fluorescence, illustrating transfection success.
Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

In Vivo Microinjection and Electroporation of Mouse Testis

This article describes microinjection and electroporation of mouse testis in&#160;vivo as a&#160;transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.

在小鼠睾丸活体显微注射和电穿孔

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...

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 ...

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.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

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 ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

一个协议来使用基因集富集分析以确定合适的动物模型转化型研究

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably identify nuclei of cardiomyocytes on histological sections, many groups rely on isolating viable cardiomyocytes prior to fixation to perform immunostaining. However, these live cardiomyocyte isolation techniques require optimization to maximize the yield, viability and quality of the samples, with inherent fluctuations from sample to sample despite maximum optimization. Here, we report a reproducible protocol, involving fixation prior to enzymatic digestion of the heart, which leads to maximum yield while preserving the in vivo morphology of individual cardiomyocytes. We further developed an automated analysis platform to determine the number of nuclei and DNA content per nucleus for individual cardiomyocytes. After exposing the chest cavity, the heart was arrested in diastole by perfusion with 60 mM KCl in PBS. Next, the heart was fixed in 4% paraformaldehyde (PFA) solution, and then digested with 60 mg/mL collagenase solution. After digestions, cells were singularized by trituration, and the cardiomyocyte fraction was enriched via differential centrifugation. Isolated cardiomyocytes were stained for Troponin T and &#945;-actinin to assess purity of the obtained population. Furthermore, we developed an image analysis platform to determine cardiomyocyte nucleation and ploidy status following DAPI staining. Image based ploidy assessments led to consistent and reproducible results. Thus, with this protocol, it is possible to preserve native morphology of individual cardiomyocytes to allow immunocytochemistry and DNA content analysis while achieving maximum yield.

The goal of this work is to develop a method to reproducibly isolate cardiomyocytes from the adult heart and measure DNA content and nucleation.

从固定心脏分离心肌细胞进行免疫细胞化学和倍体分析

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably ...