Name: in utero intra-cardiac tomato-lectin injections on mouse embryos to gauge renal blood flow
Uploaded: 2015-02-04T15:00:00.000+00:00
Duration: 10 min 25 s
Description: Watch this Scientific Journal Video about In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow at JoVE.com

The authors would like to thank Dr. George Gittes for advice and expertise throughout this study. SSL was supported by an American Heart Association fellowship (11POST7330002). Further to this SSL and this study was supported by an NIDDK Mentored Research Scientist Development Award (DK096996) and by the Children&#8217;s Hospital of Pittsburgh.

注:匹兹堡机构动物护理和使用委员会的批准大学的所有实验。 1.制备超声仪器显微注射和胚胎<ol><li>建立阶段，安装，和探针（ 图1）以及外科手术器械（ 图2）。地方的磷酸盐缓冲盐水（PBS）溶液（pH7.4）的在37℃的暖浴。完全填充微注射针与矿物油，用1ml注射器附连到第二柔性地下25针，通过其基极。 </li><li>固定针到旋转支架臂上，与矿物油溶液空针。重新填充2.5微升的TL解决方案。确保没有气泡注射针中存在。旋转针手臂朝着墙，离舞台。 </li><li>通过异氟醚持续输注麻醉孕妈妈在麻醉室。当妈妈呈现昏迷，麻醉转移到鼻管放置在C舞台audal侧，并与炉鼻的鼻管仰卧位的地方母亲以允许延续的完全麻醉状态。 </li><li>磁带肢体45°角，或手和脚休息和放置上层建筑ECG /温度显示器标签。重要的是，在怀孕的坝被连续监测，以确保麻醉足以并向眼睛软膏施加到降低干燥。 </li><li>申请脱毛产品横跨母亲的下腹部，轻轻地用干棉签擦去，然后再次用70％的乙醇饱和棉签除去任何过量的产物和头发。确保现场切口（ 图3）清洁腹部皮肤关闭所有的头发。 </li><li>执行对孕妈妈用细手术镊子和剪刀剖腹探查术。请注意，以避免削减任何重大的血管或脏器。使第一，表皮直切口1.5-2厘米以上的阴道和继续削减对肋骨的approximat伊利2-3厘米。露出皮下膜，找到白线（"白线"）（ 图3），并切平行于初始切口，沿着相同的长度，以露出内部的内脏器官和子宫saccules。 </li></ol> 2.提取胚胎<ol><li>使用6英寸棉签涂抹机动母亲和胚胎。轻轻推动（不要用力）对皮肤敷贴操纵第一胎出切口开放。提取第一胚胎球囊后，轻轻地，慢慢地，并到母亲的外部拉子宫角的其余部分。避免因切口在这个阶段拉肠或其他器官。 </li><li>量化胚胎和轻轻地放在左宫角回妈妈。然后开始把右侧子宫角回母亲开始从喇叭阴道胚胎球囊最远和向下移动。继续，直到只有两个胚胎仍然暴露（ 网络连接古尔4）。最后，在上述的柱，并平行的位置胚胎切口线，作为识别不切断子宫循环。 </li><li>放置粘土块和位置手臂和身体，以及腿和尾部之间。确保粘土表面大约级别与开腹手术切口。湿窗孔的培养皿37℃PBS。 </li><li>把握延伸钳子用右手和有孔培养皿左手（平行于胚胎开窗），并通过开窗术带来封闭镊子〜5厘米，从培养皿的顶部。打开钳完全没有撕裂开窗网。 </li><li>操纵手以上的胚胎和视线集中在这两个胚胎saccules。如果没有（或轻度）触摸saccules与开窗啮合或镊子，透过缝隙滑到他们并设置培养皿到母亲的腹部。用钳子还在延续，操作有孔的网状座位它放在一边，并在每个胚胎的基地（ 图5 </st荣&gt;）和拉镊子从切口开放。 </li><li>下一步，将装有墙培养皿中的蓝色橡胶，而不会挤压或伤害胚胎（ 图6）。确保黏土块坚决平衡培养皿中，胚胎，以及含有橡胶墙。最后，填充培养皿用37℃的PBS中直至胚完全淹没（ 图6），同时控制用于 ​​在有孔啮合泄漏。 </li></ol> 3.注射程序<ol><li>较低的注射阶段与母亲和胚胎向下使用的Z-电平调整旋钮（ 图1），然后旋转注射针及安装臂，并直接与超声波探头排队，以针半厘米的左侧和下探针尖端（ 图7） 。推针臂（未针）45°远离探头。要小心，不要打了菜或超声探头的针尖。 </li><li>提高注射阶段回到原来的高度，用超声波P长袍的正上方胚胎探头必须稍微淹没，并在3-4毫米的胚胎组织。利用X＆Y阶段调节旋钮（ 图8），修订胚/妈妈/舞台上的地位，有系统地找到跳动胚胎心脏。 </li><li>直接超声波屏幕的中心观察到黑中心目标标记绘制（*）。找到左心室（最好）或中庭采用X＆Y级调节旋钮（ 图8），然后抬高或使用旋转旋钮，在舞台上通过超声波屏幕上的黑中心标记精确定位注入目标降低的阶段。 </li><li>使用X级调节旋钮，将胚胎到右侧，关闭超声波屏。重新定位显微注射针和臂回原位，半厘米的距离和90°的超声探头（ 图7）。 </li><li>用显微注射装入调节旋钮（ 图9C-G）的位置的针与尖端清楚人igned与中心标记。调整喷射角（使用旋钮在图9C和EG），并且X，Y，和微针的Z-载体，以确保针尖被聚焦在正确的平面。仅仅使用了"注入"旋钮（ 图9F）收回显微注射针，注意不要调整其他尺寸。 </li><li>同样，使用单独的X微调阶段旋钮招回胚胎探头下，用目标正好在超声屏幕上的中心标志。确保左心室现在正好在相同的X，Y和Z平面。 </li><li>接着，只用"注入"的显微注射调节器架，慢慢穿刺胚胎与显微注射针和逐渐使前端进入左心室。注满2.5微升TL溶液进入左心室或直到空的警告提示音，通过举办"空"，挖掘"补"一次（ 图2A和B）。此时注射观察影子从它进入心脏腔室中的TL（ 图10）的针的尖端发出。相反，快速收回注射针旋转的显微注射的"注入"按钮（ 图9F）安装在注射完成。 </li><li>继续到下一个胚胎，然后重复此步骤如下这一系列的步骤剩余的胚胎，最后将胚胎最终回大坝的腹部。改用麻醉室中，轻轻移动的母亲在这里15分钟从最后一次注射时间。 </li></ol> 4.收获胚胎及分析<ol><li>通过颈椎脱位牺牲大坝。展开开腹手术切口容易地从母亲子宫中删除角。保持内子宫囊的胚胎。将在PBS冷冻胚胎。 </li><li>从解剖子宫的胚胎囊（如果进行基因分型需要收集组织），并把整个胚胎我ñ4％多聚甲醛（PFA）过夜，然后到30％蔗糖过夜，冻结在低温恒温器切片中。然后切冷冻切片，并将其放置到幻灯片免疫组化分析。任选地，使用的组织为通过脱水在100％的甲醇，在4％PFA以下定影整个安装。 </li><li>免疫组化分析，将进入冰冻切片PBS去除十月然后方框，用10％正常驴血清的部分30分钟。然后添加PECAM一级抗体以1:100稀释过夜，在4℃。第二天，洗涤载玻片中的PBT 3次，每次10分钟，并添加驴抗鼠594仲和孵育1小时，在室温。 注:这是一个非常苛刻的技术，需要超和显微注射的优化和协调。有一个非常陡峭的学习曲线，与此相关的技术，需要四到六周注射辅导就会有一个精通之前。 </li></ol>

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	<li>Abrahamson, D. R., Robert, B., Hyink, D. P., St John, P. L., Daniel, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+and+formation+of+microvasculature+in+the+developing+kidney.">Origins and formation of microvasculature in the developing kidney.</a> Kidney international. Supplement. 67, S7-S11 (1998).</li><li>Sims-Lucas, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Progenitors+Exist+within+the+Kidney+and+Lung+Mesenchyme.">Endothelial Progenitors Exist within the Kidney and Lung Mesenchyme.</a> PloS one. 8 (6), e65993 (2013).</li><li>Dressler, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+early+kidney+specification,+development+and+patterning.">Advances in early kidney specification, development and patterning.</a> Development. 136 (23), 3863-3874 (2009).</li><li>Costantini, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+controls+and+cellular+behaviors+in+branching+morphogenesis+of+the+renal+collecting+system.">Genetic controls and cellular behaviors in branching morphogenesis of the renal collecting system.</a> Wiley Interdiscip Rev Dev Biol. 1 (5), 693-713 (2012).</li><li>Costantini, F., Kopan , R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+a+complex+organ:+branching+morphogenesis+and+nephron+segmentation+in+kidney+development.">Patterning a complex organ: branching morphogenesis and nephron segmentation in kidney development.</a> Dev Cell. 18 (5), 698-712 (2010).</li><li>Das, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal-epithelial+crosstalk+regulates+kidney+progenitor+cell+differentiation.">Stromal-epithelial crosstalk regulates kidney progenitor cell differentiation.</a> Nat Cell Biol. 15 (9), 1035-1044 (2013).</li><li>Rymer, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+blood+flow+and+oxygenation+drive+nephron+progenitor+differentiation.">Renal blood flow and oxygenation drive nephron progenitor differentiation.</a> Am J Physiol Renal Physiol. , (2014).</li><li>Shah, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+mouse+blood+flow+and+oxygen+correlate+with+early+pancreatic+differentiation.">Embryonic mouse blood flow and oxygen correlate with early pancreatic differentiation.</a> Developmental biology. 349 (2), 342-349 (2011).</li><li>Andres, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EphB4+receptor+tyrosine+kinase+transgenic+mice+develop+glomerulopathies+reminiscent+of+aglomerular+vascular+shunts.">EphB4 receptor tyrosine kinase transgenic mice develop glomerulopathies reminiscent of aglomerular vascular shunts.</a> Mech Dev. 120 (4), 511-516 (2003).</li><li>Wagner, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+imaging+of+kidney+vascular+corrosion+casts+with+Nano-CT.">High-resolution imaging of kidney vascular corrosion casts with Nano-CT.</a> Microsc Microanal. 17 (2), 215-219 (2011).</li></ol>

The authors declare that they have no competing financial interests.

注射麻醉和时间框架与关于母亲的麻醉，这是至关重要的，以保持气流不变（2-3升/分钟），并在低的PSI。镇静药流必须在约1.75-2升/分举行。同时，时间安排在注射发生必须密切监测和控制与每个垃圾。对于每一个垃圾注射过程应保持在45分。这一时限是极为重要的实验，因为每个胚胎必须保持恒定，正常的心脏速率的范围内，以促进在整个身体和感兴趣器官受控和恒定的灌?...

在胚胎发育两个分立的，但同时，血管过程发生:血管生成，该过程从一个主要的预先存在的血管，和血管生成，这是一种新形成血管的来自住宅内皮祖1,2-由此容器增长。分别，前者是同义的血流量，而后者被认为主要是发生在没有它。 同时，以血管形成，肾脏祖细胞的合成，增殖和分化周期性和动态的过程开始展开上9.5天胚胎（E9.5）。在这一点上输尿管芽（UB）侵入背侧到包围后肾间质（MM），并继续直到出生3。反复分支UB进入快速冷凝后肾间质帽开始肾脏的功能单位，肾的形成。随着每一代新UB和NEPH的罗恩，老一辈流离失所到内皮层和髓质的地区，在那里他们再进行内主要血管密集的环境中进一步成熟和分化。就证明了德雷斯勒等人 3，此胚胎过程是由感应信令沉淀，如UB和MM，和细胞外因素3-6无数之间的串扰。最近的两个研究发展胰腺内的细胞外因素和肾脏包括氧张力和血流量7,8。后者将在更详细地相对于肾脏发育中讨论如下。 以暴露所述感应作用血流可能起着肾祖细胞的分化，以及在其他器官的过程，胚胎血流映射的精确和准确的方法是必要的。 计量血流的替代方法包括微升的处方trasound成像及树脂注塑9,10。决定性，这些模式已经被证明是固有地缺乏其能力，以同时地揭开血流之间的时间和空间的并置和干细胞的分化。树脂管型，例如，提供成人组织中血管形成图案的一个有效的模型，然而，在未成熟的血管，如与胚胎的时间点，容器是非常不发达和漏。因此，树脂注塑未能微小的，常常多孔，船内举行。 对于这些明显的障碍，除其他外，我们选择了把超声引导体内心内胚胎番茄凝集素（TL）显微注射到我们的肾发展的调查工作。在此过程中，我们利用超声波探头向同步引导装入微量针填充有2.5微升的TL溶液注入小鼠胚胎在E11.5，E13.5，E15.5和E17.5的时间点左心室。 E170.5是最新的发育年龄为针是没有强大到足以穿透更发达的胚胎。 这种显微注射方法的优点是丰富的。超声引导下注射允许注射针的胚胎左心室内精确定位，被动和控制驱逐溶液进入动物，对心脏的损害最小和周围组织的心脏跳动，并避免突发性心脏衰竭而死亡胚胎前全身灌注。与使用FITC标记TL的，任何灌注脉管将保持沿着其内皮顶膜的标志物。与免疫相结合，利用粘附分子（CD31，血小板内皮细胞粘附分子）和其他各种血管标记，我们能够清楚地灌注和未灌注血管区分，以及表征周围组织中的任何异常染色。

<table><tbody><tr><td>DAPI</td><td>Sigma Aldrich</td><td>022M4004V</td><td>concentration 1:5,000</td></tr><tr><td>Pecam</td><td>BD Biosciences</td><td>553370</td><td>concentration 1:100</td></tr><tr><td>FITC-Tomato Lectin</td><td>Vector Laboratories</td><td>FL-1321</td><td>concentration 2.5 &amp;micro;l / embryo</td></tr><tr><td>Alexa Fluor-594 (Donkey Anti-Rat)</td><td>Jackson Immunoresearch</td><td>712-585-150</td><td>concentration 1:200</td></tr><tr><td>Microinjection Needle</td><td>Origio Mid Atlantic Devices</td><td>C060609</td><td></td></tr><tr><td>Mineral Oil</td><td>Fisher Scientific</td><td>BP26291</td><td></td></tr><tr><td>1 ml syringe</td><td>Fisher Scientific</td><td>03-377-20</td><td></td></tr><tr><td>Clay Blocks</td><td>Fisher Scientific</td><td>HR4-326</td><td></td></tr><tr><td>Surgical Tape</td><td>Fisher Scientific</td><td>18-999-380</td><td></td></tr><tr><td>PBS</td><td>Fisher Scientific</td><td>NC9763655</td><td></td></tr><tr><td>Hair Removal Product</td><td>Fisher Scientific</td><td>NC0132811</td><td></td></tr><tr><td>Surgical Scissors</td><td>Fine Science tools</td><td>14084-08</td><td></td></tr><tr><td>Fine Forceps</td><td>Fine Science tools</td><td>11064-07</td><td></td></tr><tr><td>Surgical Marking Pen</td><td>Fine Science tools</td><td>18000-30</td><td></td></tr><tr><td>Right angle forceps (for hysterectomy)</td><td>Fine Science tools</td><td>11151-10</td><td></td></tr></tbody></table>

in utero intra-cardiac tomato-lectin injections on mouse embryos to gauge renal blood flow

形成和发展肾脏血管（除了肾小球）灌注大大充分研究。作为脉管经由血管生成的发展（其为分支大血管的）和血管（ 从头血管形成），如树脂管型， 在体内超声成像，和显微解剖灌注映射技术在证明之间的亲密关系被限制胚胎内这两个过程和显影肾结构。在这里，我们描述了在小鼠胚胎在子宫内的心内超声引导FITC标记番茄凝集素显微注射的方法，以评估肾灌注的个体发育。番茄凝集素（TL）被灌注在整个胚胎和肾脏收获。组织中共同染色关于各种肾结构，包括:肾祖细胞，肾单位的结构，输尿管上皮细胞，和血管。起始于E13.5大口径血管灌流，但是周边船只仍unperfused。由E15.5和E17.5，小末梢血管以及肾小球开始变得灌注。这个实验技术对于胚胎发育过程中学习脉管和血流的作用是至关重要的。

血管的形成先于肾脏发育流大多数胚胎组织（包括肾脏）中包含的致密脉管（包括unperfused和灌注），即使是在早期胚胎的时间点。到肾脏发育中更好地衡量和分析血流我们利用在宫内胚胎心内显微注射的方法。与使用一个高分辨率超声在E11.5通过E17.5识别胚胎心脏，并按照通过剖腹提取和单一子宫球囊的曝光，我们能够注入2.5微升TL的（异硫氰酸荧光素素（FITC）缀合）。 <p...

Watch this Scientific Journal Video about In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow at JoVE.com

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

This manuscript describes a technique for visualization of the developing vasculature. Here we utilized in utero intra-cardiac FITC-labeled tomato lectin microinjections on mouse embryos. Using this technique, we delineate the perfused and unperfused vessels throughout the embryonic kidney.

在对小鼠胚胎子宫内心脏番茄凝集素注射到计肾血流量

The formation and perfusion of developing renal blood vessels (apart from glomeruli) are greatly understudied. As vasculature develops via angiogenesis ...

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In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

9.7K 次观看.  Children's Hospital of Pittsburgh of UPMC. This manuscript describes a technique for visualization of the developing vasculature. Here we utilized in utero intra-cardiac FITC-labeled tomato lectin microinjections on mouse embryos. Using this technique, we delineate the perfused and unperfused vessels throughout the embryonic kidney.

视频: In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

一个小鼠模型在子宫内移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

科研

医学

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or &#945;v&#946;3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

对比成像的小鼠胚胎使用高频超声

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

发育生物学

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

一种新的手术方法气管内注射生物活性剂在胎鼠模型

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Murine Fetal Echocardiography

Transgenic mice displaying abnormalities in cardiac development and function represent a powerful tool for the understanding the molecular mechanisms underlying both normal cardiovascular function and the pathophysiological basis of human cardiovascular disease. Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development 1-3. In order to study the role of genetic or pharmacologic alterations in the early development of cardiac function, ultrasound imaging of the live fetus has become an important tool for early recognition of abnormalities and longitudinal follow-up. Noninvasive ultrasound imaging is an ideal method for detecting and studying congenital malformations and the impact on cardiac function prior to death 4. It allows early recognition of abnormalities in the living fetus and the progression of disease can be followed in utero with longitudinal studies 5,6. Until recently, imaging of fetal mouse hearts frequently involved invasive methods. The fetus had to be sacrificed to perform magnetic resonance microscopy and electron microscopy or surgically delivered for transillumination microscopy. An application of high-frequency probes with conventional 2-D and pulsed-wave Doppler imaging has been shown to provide measurements of cardiac contraction and heart rates during embryonic development with databases of normal developmental changes now available 6-10. M-mode imaging further provides important functional data, although, the proper imaging planes are often difficult to obtain. High-frequency ultrasound imaging of the fetus has improved 2-D resolution and can provide excellent information on the early development of cardiac structures 11.

Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development. High-frequency ultrasound imaging has improved 2-D resolution and can provide excellent information on early cardiac development and is an ideal method to detect the impact on cardiac structure and function prior to death.

小鼠胎儿超声心动图

Transgenic mice displaying abnormalities in  cardiac development and function represent a powerful tool for the understanding  the molecular mechanisms ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

心率在小鼠中通过无创M型超声心动图测量子宫内

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment.

小鼠宫内移植中静脉和羊膜内植入的研究

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

小鼠脑脊髓子宫转导后超微结构神经可塑性参数的评价

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

全山荧光在小鼠胚胎发生过程中心脏室发育的分析

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

β-aminopropionitrile 诱发主动脉瘤和解剖小鼠模型 Aortopathy 的定量显微 CT 分析

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

细胞标记和注射胚胎发展小鼠心脏

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...