这项工作是支持由授予的MRC到M FS。

1。胚胎的发展<ol><li>当他们被解雇，鸡蛋都聚集由于绒毛膜附件细丝。为了让胚胎发育正常，有必要单独的鸡蛋。昆布和削减附件细丝，两个钳附件细丝。 </li><li>后unclustering，鸡蛋是从粪便和藻类分离和转移的最大密度40％6厘米培养皿鸡蛋新鲜胚胎培养基。 </li><li>青鳉胚胎发育略慢比斑马鱼在27 ° C。孵化的时间是不同的两个物种之间;鳉胚胎孵化7天绒毛膜，并立即开始游泳，吃饭，而斑马鱼的胚胎在2天的孵化，但开始游泳，吃5-6天。青鳉发展是阶段性的，根据岩松的分期 4 。 </li><li>青鳉胚胎的发展，可以很方便地调整实验计划，选择适当的温度。在4 ° C可以停止发展鳉胚胎处于早期开发阶段。阶段24后心脏跳动启动时，发展可减缓使用最低气温18 ° C。 </li><li>器官/组织出现的时机是在青鳉与斑马鱼相比略有不同，即在青鳉somitogenesis发生后大脑发育的开始，而在斑马鱼somitegenesis大脑发育之前。 </li></ol> 2。卸下绒毛膜鳉绒毛膜由两个保护层，用硬的内层和外表面软。因此，采用链霉蛋白酶和孵化酶的两个步骤蛋白酶治疗是必要删除此绒毛膜。 一旦dechorionated，胚胎应保持在1X基站。半无菌条件下，将加强dechorionated胚胎的成功文化，特别是当需要较长时期的观察。这些措施包括使用无菌解决方案（如消毒用抗生素1X BSS）和70％的乙醇与1X基站冲洗消毒的工具。 由于dechorionated鳉胚胎比dechorionated斑马鱼的胚胎更柔和，脆弱，格外小心，必须采取措施，确保他们不接触空气或气泡中的吸管，因为这将导致立即崩溃。要确保到胚胎的损伤最小，应该是一个广口热抛光的玻璃吸管，吸管泵用于转移胚胎和头发循环应当利用定位观察胚胎。非贴壁的培养皿中，应使用，以防止胚胎附着在表面。 <ol><li>在来dechorionation之前，应当检查，鸡蛋已充分分离和清理。 </li><li>由于链霉蛋白酶和孵化酶，蛋白酶，它们应保持在冰上，并尽量减少接触到这些酶的胚胎。 </li><li>将鸡蛋砂纸（P2000粒度，防水）放置在一个9厘米培养皿的盖子。删除多余的介质，但保证足够的量仍然以防止干燥胚胎。 </li><li>轻轻滚45-60秒左右的胚胎，以消除一些外表面的毛发，轻轻得分绒毛膜表面（图1）。回到原来的培养皿转移胚胎和检查。 砂纸轧胚时用食指，用最小的压力，保持手指平行面沙纸。不要卷超过一次5-7胚胎。这种预防措施，将最大限度地减少破碎下对方的胚胎的风险。 </li><li>替换盘20mg/ml为40-60分钟的链霉蛋白酶和孵化的胚胎蛋中等27 ° C。 确保胚胎有足够的覆盖链霉蛋白酶和盖菜。未使用的链霉蛋白酶应保持在冰上，在这一步，以尽量减少自我消化，可以重复使用，直到活动失去（约1-2周）。 </li><li>恢复重用和洗胚胎，在胚胎中期的5倍，以消除链霉蛋白酶的痕迹，因为这将可灭活该孵化酶添加链霉蛋白酶。 </li><li>取出胚胎孵化酶介质和覆盖胚胎，以确保胚胎坐在单层菜。 如果鸡蛋在彼此之上坐的菜，那些在底部会被压碎蛋壳溶解。未使用的孵化酶置于冰上。 </li><li>孵育胚胎在27℃，15分钟后，定期检查使用立体显微镜的孵化进度。 它将会看到月球火山口状孔开始出现在绒毛膜内层，很快溶解以下这使软外层的蛋壳容易去除手册册Y.整个孵化过程可能需要15-60分钟。 </li><li>作为胚胎绒毛膜来自胚胎转移到培养皿中含有1X的BSS。 确保不转让触摸到表面的BSS允许胚胎轻轻铺开吸管尖孵化酶的1X的BSS。 </li><li>一旦所有的胚胎孵化酶的转移，最终转移到另一个1X BSS的新鲜菜。 这可以确保胚胎不会受到任何残存的孵化酶，因为这将损害暴露胚胎。一旦在这最后一盘的任何胚胎仍拥有绒毛膜外层可手动解放使用的体视显微镜下消毒，钳微。 如果胚胎需要制定以下dechorionation，应添加青霉素/链霉素的BSS，以防止细菌生长。 </li></ol><img src="/files/ftp_upload/2055/2055fig1.jpg" alt="图1" /> 图1。dechorionation卷起和展开的胚胎比较。观察如何在B组中的热轧胚胎缺乏面板A.展开胚胎的头发 3。挂载dechorionated胚胎较长时期的成像活胚胎（如时间推移成像）以及固定胚胎的详细观察，琼脂糖包埋是有用的。在原肠早期器官（阶段14日至28阶段），青鳉胚胎表现出的海浪有节奏的收缩运动，组织胚胎发育和卵黄 5层覆盖整个周皮。胚胎是用3.5mm的1 -庚停止收缩运动6。 要停止运动阶段28（64hpf）后的胚胎，胚胎是麻醉之前加入嵌入滴Tricaine磺酸（TMS）。 TMS也加入到琼脂糖（只添加少量，这个剂量，需要进行优化，大约1:25稀释）。 <ol><li>少量的3％的低胶凝温度琼脂糖（1X BSS）通过加热至37℃，并保持在此温度下解冻。 </li><li>下面的步骤需要迅速进行，以确保琼脂糖不巩固前定向胚胎。使用宽口的玻璃吸管转移，足以熔化琼脂糖，以填补Eppendorf管中的第，抑郁症。 </li><li>一个dechorionated胚胎转移到第抑郁症，最大限度地减少以上的BSS进行胚胎。立即吸收琼脂糖和胚胎从第抑郁症和转移到培养皿中文化室。 一个3.5厘米在底部的玻璃窗口的Petri菜。对于使用倒置显微镜成像，胚胎导向的身体面朝下放置在靠近玻璃盖。对于使用一个堂堂正正的显微镜成像，琼脂糖厚度降到最低。 </li><li> 3.5厘米培养皿转移到一个14厘米直径的Petri含有冰和水（装满水的总深度大约1 / 3的大型盘）的菜。虽然坚持14cm的培养皿底部的上下腔，使用吹风循环轻轻定位在熔化的琼脂糖所需的胚胎保存的胚胎，直到琼脂糖轻轻的提高和降低较小的菜到原来的位置，巩固在冰冷的水中。 </li></ol> 4。在青鳉胚胎的细胞移植此过程的目标是，以确定是否感兴趣的基因细胞自主（一个单元格内），或者非细胞自主细胞之间的行为。 供体细胞标记示踪染料如罗丹明-葡聚糖移植前（如在陪同朱庇特议定书"青鳉胚胎显微注射"中所述），或可利用与绿色荧光蛋白表达的转基因株，使移植的评估。两种标记技术的组合往往是有用的，以克服可能遇到的自体荧光。移植后的时间推移研究，GFP的表达是非常有用的。 其次是使用一个宽口的玻璃吸管，用吸管泵的整个过程中所有的工具（包括幻灯片）事先用70％乙醇消毒彻底无菌1X BSS冲洗。收件人的胚胎通常发展阶段12左​​右，因为这允许的腹侧和背侧的两极时进行移植歧视。 <ol><li>开始dechorionating胚胎移植注射器具和酶孵化期间设置之前1.5小时。 </li><li>成9厘米直径培养皿中放置一个腔显微镜幻灯片。 </li&gt; <li>添加少量的3％甲基纤维素腔使用无菌枪头的幻灯片中心和分布得很散。 </li><li>干甲基大约1-2分钟。 </li><li>加入350μl的无菌1X BSS，以填补幻灯片抑郁症。 </li><li>一个捐赠者的胚胎和四个收件人胚胎转移到幻灯片中使用的玻璃吸管。 </li><li>头发循环​​使用，使胚胎的胚盘是朝上的定位胚胎。 胚胎可以靠在互相反对进一步增加稳定。 </li><li>轻轻插入捐助胚，慢慢地摄取10-20细胞微针。 </li><li>轻轻插入收件人胚胎胚所需面积的针和驱逐细胞缓慢。应采取十分谨慎，当插入收件人的胚盘细胞，从而不破坏细胞卵黄囊边界，因为这将导致胚胎死亡。 </li><li>倒入培养皿消毒1X BSS。 小心倒入碟的BSS尽可能靠近一侧的菜，以免破坏胚胎。切勿直接倒入BSS到胚胎，并添加足够的BSS，幻灯片和胚胎都沉浸。 </li><li>添加青霉素/链霉素100μL的菜和覆盖。小心地转移菜到27 ° C培养箱允许的正常发展。 </li><li>所需的胚胎可以定期观察。当使用移植进行增益功能和/或表型救援实验，观察到的一些形态学改变可能是由于移植的影响。因此，多个器官移植是必要的。 melanophores要经过2-3天，卵黄囊，头部，眼睛和树干上。如果目前对移植胚胎，然后一个成功的嵌合体有最有可能被生产（图3）。 </li></ol>如果必要，基因型捐助转移到PCR管（S），其中包含55 °为4小时10分钟之后在94℃，进行PCR扩增。25μl20mg/ml蛋白酶K孵化的胚胎（S） 5。代表性的成果<img src="/files/ftp_upload/2055/2055fig2.jpg" alt="图2" /> 图2。琼脂糖嵌入式胚胎的位置。前路右侧所示的看法是背。 B组:内皮细胞可以看出胚胎体两侧和使用FLI启动子驱动的GFP标记。 <img src="/files/ftp_upload/2055/2055fig3.jpg" alt="图3" /> 图3移植后胚胎的图片。图像立即显示了一个捐赠者和接受者的胚胎移植后。显示在右边的捐助胚胎是一个完全的红色胚（箭头）。收件人的胚胎是显示在左侧，很容易被确定的红色小肿块可见移植细胞胚（箭头）。图片B显示两个收件人胚胎移植后（在27 ° C孵育）约7小时。注意移植的细胞移植部位迁移，并正在通过胚（箭头）分散。图片C显示在收件人胚胎st.29（约74hpf）。注意色素沉着的眼睛，躯干和大脑区域（箭头）的melanophores存在。罗丹明标记的细胞可以被看作是殖民表明一个嵌合体的成功生产的胚胎结构。

<ol>
	<li>Furutani-Seiki, M., Sasado, T., Morinaga, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+genome-wide+screen+for+mutations+affecting+organogenesis+in+Medaka,+Oryzias+latipes.">A systematic genome-wide screen for mutations affecting organogenesis in Medaka, Oryzias latipes.</a> Mech Dev. 121, 647-658 (2004).</li><li>Furutani-Seiki, M., Wittbrodt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medaka+and+zebrafish,+an+evolutionary+twin+study.">Medaka and zebrafish, an evolutionary twin study.</a> Mech Dev. 121, 629-6237 (2004).</li><li>Hirose, Y., Varga, Z. M., Kondoh, H., Furutani-Seiki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+lineage+and+regionalization+of+cell+populations+during+Medaka+neurulation.">Single cell lineage and regionalization of cell populations during Medaka neurulation.</a> Development. 131, 2553-2563 (2004).</li><li>Iwamatsu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+normal+development+in+the+medaka+Oryzias+latipes.">Stages of normal development in the medaka Oryzias latipes.</a> Zool Sci. 11, 825-839 (1994).</li><li>Cope, J., Fluck, R., Nicklas, L., Plumhoff, L. A., Sincock, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stellate+layer+and+rhythmic+contractions+of+the+Oryzias+latipes+embryo.">The stellate layer and rhythmic contractions of the Oryzias latipes embryo.</a> J Exp Zool. 254, 270-275 (1990).</li><li>Brend, T., Holley, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+Whole+Mount+High-Resolution+Double+Fluorescent+In+Situ+Hybridization.">Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization.</a> J Vis Exp. , (2009).</li></ol>

在这个视频中，我们将演示如何dechorionate开展鳉胚胎细胞transplanataion。这是一个强大的技术，生产用于在开发过程中的基因/蛋白功能的研究，以及阐明了有问题的基因/蛋白质的自主权的嵌合胚胎。青鳉是一个有用的脊椎动物模型系统行之有效的命运地图和其透明度贷款，它们在体内的实时成像，由于这种技术。移植可结合显微注射（如在陪同朱庇特议定书"青鳉胚胎显微注射"所述），因此可以很容易地移植细胞的行为观察。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Embryo medium</td>
<td>Reagent</td>
<td>Made in-house</td>
<td>N/A</td>
<td>200ml 50X stock solution, 1ml 1%methyleneblue in H2O/10 liter RO water (50X stock solution: NaCl 14.7g, KCl 0.6g, CaCl2 2H2O 2.4g, MgSO4 7H2O 4.0g/1 litre RO water.)</td>
</tr>
<tr>
<td>Micro-dissecting forceps</td>
<td>Tools</td>
<td>55 INOX A.DUMONT&amp;FILS</td>
<td>N/A</td>
<td>Very sharp fine tips for removal of chorion.</td>
</tr>
<tr>
<td>Fine waterproof sandpaper - p2000 grit size</td>
<td>Tools</td>
<td>Hermes Abrasives Ltd.</td>
<td>N/A</td>
<td>Very fine grit size for rolling/cleaning embryos with chorions.</td>
</tr>
<tr>
<td>3% methylcellulose</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M 0512</td>
<td>High viscosity for holding embryos in place during transplantation.</td>
</tr>
<tr>
<td>1X BSS</td>
<td>Reagent</td>
<td>Made in-house</td>
<td>N/A</td>
<td>20XBSS: 130g NaCl, 8g KCl, 4g MgSO4.7H2O, 4g CaCl2.2H2O, and 10mg Phenol Red in 1L MilliQ water and autoclave; 500mM Hepes in MilliQ water autoclaved. Add 25ml 20XBSS and 15ml 500ml Hepes pH7.0, fill up to 500ml and filter sterilize before use.</td>
</tr>
<tr>
<td>Penicillin-streptomycin</td>
<td>Reagent</td>
<td>Gibco</td>
<td>15140-122</td>
<td>Added to BSS when incubating dechorionated embryos.</td>
</tr>
<tr>
<td>Pronase</td>
<td>Reagent</td>
<td>Calbiochem</td>
<td>53702</td>
<td>For dechorionation.</td>
</tr>
<tr>
<td>Hatching enzyme</td>
<td>Reagent</td>
<td>Made in-house</td>
<td>N/A</td>
<td>For dechorionation.</td>
</tr>
<tr>
<td>Tricaine mesilate (TMS)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-5040</td>
<td>400mg tricaine in 97.9ml distilled water and 2.1ml 1M Tris, pH adjusted to 7; 4.2ml of this solution in 100ml clean tank water.</td>
</tr>
<tr>
<td>3% ultra-low gelling temp. agarose</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-2576</td>
<td>Type IX-A. Used during embedding of embryos for observation.</td>
</tr>
<tr>
<td>Petri dish culture chamber</td>
<td>Tool</td>
<td>Iwaki, Asahi Techno Glass Corp.</td>
<td>11-004-008</td>
<td>Iwaki glass base dish 35mm with 12mm window. Used during embedding of embryos for observation.</td>
</tr>
<tr>
<td>Micromanipulator</td>
<td>Equipment</td>
<td>Narishige</td>
<td>N/A</td>
<td>Model MN151. For removal and insertion of cells during transplantation.</td>
</tr>
<tr>
<td>Borosilicate glass capillaries</td>
<td>Tool</td>
<td>Harvard Apparatus</td>
<td>GC100-10T</td>
<td>For making transplantation needles.</td>
</tr>
<tr>
<td>Flaming/Brown micropipette puller</td>
<td>Equipment</td>
<td>Sutter Instrument Co.</td>
<td>Model P-97 </td>
<td>For making transplantation needles.</td>
</tr>		</tbody>
		</table>
		</div>

dechorionation of medaka embryos and cell transplantation for the generation of chimeras

青鳉是一个小产蛋的淡水鱼类，允许遗传和胚胎学分析，是在表型驱动的全基因组突变的屏幕进行了1生物体的脊椎动物模型之一。鳉和斑马鱼之间的相关基因的功能重叠的分歧使得新颖，品种单一，从而鳉和斑马鱼是脊椎动物的基因组功能基因清扫互补难以辨认的表型鉴定。青鳉胚胎，如dechorionation，安装成像和细胞移植的胚胎，操纵鳉和斑马鱼在实验室工作的主要程序。细胞移植研究细胞鳉突变的自主权。嵌合体产生的未标记的收件人胚胎移植到从捐赠的胚胎标记细胞。供体细胞可以移植到特定领域基于命运的地图3上，使移植细胞克隆可以集成在开发过程中的利益的组织中的收件人胚胎。由于硬的蛋壳和软的胚胎，操纵鳉胚胎是比在斑马鱼中更多地参与。在这个视频中，我们展示操纵鳉胚胎的详细程序。

Watch this Scientific Journal Video about Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras at JoVE.com

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

由于硬的蛋壳和软的胚胎，操纵鳉胚胎是比在斑马鱼中更多地参与。该视频显示一步一步的程序，包括dechorionation如何操作鳉胚胎，在琼脂糖凝胶成像和细胞移植嵌合体的生产安装。这些程序是必不可少的，在实验室中使用鳉和斑马鱼为脊椎动物的基因组功能基因清扫充分利用其互补性的特点。

鳉胚胎和细胞移植嵌合体生成Dechorionation

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in ...

dechorionation-medaka-embryos-cell-transplantation-for-generation

Nanyang Technological University

Research

JoVE Journal

Biology

17.6K 次观看.  University of Bath. 由于硬的蛋壳和软的胚胎，操纵鳉胚胎是比在斑马鱼中更多地参与。该视频显示一步一步的程序，包括dechorionation如何操作鳉胚胎，在琼脂糖凝胶成像和细胞移植嵌合体的生产安装。这些程序是必不可少的，在实验室中使用鳉和斑马鱼为脊椎动物的基因组功能基因清扫充分利用其互补性的特点。

视频: Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

分离和造血干细胞移植（造血干细胞）

科研

生物学

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

6.5 DPC小鼠胚胎剖析

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

Tracheotomy: A Method for Transplantation of Stem Cells to the Lung

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell therapy may alleviate many of the symptoms associated with lung disease and induce alveolar repair in adults. Concurrent with the ongoing search for stem cells applicable for human treatment, precise delivery and homing (to the site of disease) must be reassured for successful therapy. Here, I report that stem cells can safely be instilled via the trachea opening a non-stop route to the lung. This method involves a skin incision, caudal insertion of a cannula into and along the tracheal lumen, and injection of a stem cell vehicle mixture into airways of the lung. A broad range of media solutions and stabilizers can be instilled via tracheotomy, resulting in the ability to deliver a wider range of cell types. With alveolar epithelium confining these cells to the lumen, lung expansion and negative pressure during inhalation may also assist in stem cell integration. Tracheal delivery of stem cells, with a quick uptake and the ability to handle a large range of treatments, could accelerate the development of cell-based therapies, opening new avenues for treatment of lung disease.

Significant breakthroughs in stem cell identification are continuously being made. To translate these discoveries, however, novel methods for cellular delivery must be devised. Here I report that the airways provide a safe route for stem cell transplantation to the lungs.

气管切开术：干细胞移植肺的方法

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell ...

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

塑料嵌入和非洲爪蟾胚胎切片

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

人CD40激活的B细胞的生成

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

移植嵌合斑马鱼胚胎生成

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

组织有针对性的胚胎嵌合体：斑马鱼原肠胚细胞移植

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

电子显微镜对胚胎的制备果蝇胚胎心管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

移植绿色荧光蛋白表达对视网膜和大脑发育的实时成像嵌合斑马鱼胚胎的卵裂球

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

作为一个型号的遗传生物鳉胚胎显微注射

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...