这项工作是由来自美国国立卫生研究院的研究补助金（HL068915 HL088206），从长尾研究基金的研究员奖，并在波士顿儿童医院的心脏传导基金捐款支持。

第1部分：组装构建压铸模具<ol><li>使用刀片硅胶管（VWR）减半，切成3厘米长的片。 </li><li>油管两端的内侧放置一个植入级RTV硅酮胶（罗地亚）下降。 </li><li>快速放置小块（1厘米× 1厘米）的聚酯丝网（麦克马斯特CARR）上的硅酮胶下降，并配合管端。这将提供一个微微凸起，平坦的表面构造附件。重复的另一端。 </li><li>允许模具在室温下干燥3天。一次20到30的模具是非常有用，因为这些基本上是一次性的。 </li><li>胶粘剂完全干燥后，储存在充满70％乙醇的烧杯，直到他们需要这些模具。这一步，​​将有助于消毒模具及成品，如图1所示。 </li></ol>第2部分：成肌细胞分离的制备<ol><li>稀释1毫克层粘连蛋白（Sigma公司）在250毫升0.22微米过滤灭菌的磷酸盐缓冲液（PBS）含有1％青霉素/链霉素（Invitrogen公司）和1％Fungizone（Invitrogen公司）。 </li><li>大衣150毫米组织培养皿（BD猎鹰）4步骤1.1 mL的稀释层粘连蛋白的解决方案和电镀前主骨骼肌成肌细胞在37℃孵育至少4个小时。 </li><li>混合20％胎牛血清（亚特兰大生物），5毫微克/升基本的成纤维细胞生长因子（Promega公司），1％青霉素/链霉素（Invitrogen公司），和1％Fungizone火腿的F - 10营养合剂（Sigma公司）的成肌细胞培养基（ Invitrogen公司）。 </li><li>溶解1克〜295 U /毫克胶原酶2 100毫升2.4 U / mL的Dispase - 2（罗氏）（顿），准备骨骼肌消化解决方案。添加0.037克氯化钙2拌匀。通过使用一个0.2微米的过滤器在-20 ° C到10毫升的注射器和存储等分装的磁盘的过滤消毒消化缓冲将组织消化在37 ° C，与成肌细胞媒体所需的数额。 </li></ol>第3部分：从骨骼肌成肌细胞隔离<ol><li>使用镊子和小剪刀，除去死亡新生儿的Lewis大鼠椎旁肌肉切片沿脊髓背下来，脱皮的皮肤和筋膜层，然后切除的肌肉。 </li><li>在组织培养罩，置于50 mL锥形管40毫升的1X汉克斯平衡盐溶液（HBSS）冰（Invitrogen）的填充（BD猎鹰）切除的肌肉。允许肌条定居到试管底部，小心地取出，用无菌的巴斯德吸管在文化遮光罩（贝克），真空吸液和血液残余。 </li><li>到150毫米的培养皿中倒入收获的肌肉块，并删除剩余的液体吸可能。使用2个单刃刀片直言不讳地组织到稠糊状。预热消化解决方案，并倒上粘贴的混合物转移到一个新的50 mL试管，使用10毫升吸管。在酶消化，偶尔磨碎组织（不冒泡）10毫升装到一个无绳移液器援助（德拉蒙德）吸管。再次，让组织来解决和消除多余的液体。 </li><li>广场上的摇摆平台管和消化约30分钟，在37 º C。 </li><li>肌肉一旦液化，通过在室温下10分钟600 XG（美国贝克曼GP）的解决方案，通过一个70微米的细胞过滤器（BD猎鹰）和离心机。用巴斯德吸液管和吸，尽可能多的液体尽可能消除和10毫升温暖的成肌细胞培养基重悬沉淀。 </li><li>板前未涂层的150毫米15分钟的组织培养板的细胞，以帮助消除成纤维细胞。 </li><li>转让尚未连接层粘连蛋白涂层cultureware和稀释，以便有一个板块为每1至2幼鼠的细胞。在37℃，5％CO 2，放置在一个培养箱集板。 </li></ol>第4部分：铸造工程的组织结构（5毫升） <ol><li>经过1〜2天，取出从孵化器的150毫米的成肌细胞组织培养板10，37℃1X PBS冲洗两次，并添加4毫升温暖的0.05％胰蛋白酶EDTA每块板。返回板块的孵化器，为5分钟。 </li><li>收获在文化罩板分离成肌细胞，并收集在50毫升管的液体（BD猎鹰）。成肌细胞培养基用10毫升冲洗所有的板块，并结合在离心管中的细胞。颗粒细胞在室温为10分钟600 XG（美国贝克曼GP），除去多余的液体，在步骤3.5中所述。 </li><li>成肌细胞重悬在1.6毫升的成肌细胞的培养基，并置于冰上。 </li><li>使用镊子，小心地取出1.5步骤4构造的模具，用1X PBS冲洗两次。关闭所有与巴斯德吸管的PBS的痕迹，然后放置在一个6孔组织立方米每个模具的真空吸lture板（BD猎鹰）。 </li><li>上冰收集的其他建设准备所需的试剂（10X火腿的F10键，抗生素，1型胶原[3.9毫克/毫升] [6]和基质胶™），并保持在37℃的成肌细胞的媒体联合收割机500μL，10X火腿的F10键，100μL青霉素/链霉素，100μLFungizone，1.6毫升的胶原蛋白，在15 mL锥形管和750μL基质胶™。 10秒使用小型Vortexer（VWR）设置为10和地点上的冰混合的解决方案。 </li><li>接下来，添加80μL碳酸氢钠3和步骤3.3中的成肌细胞悬液的混合物从步骤3.5。涡整个混合物为10秒，返回管冰桶，几分钟，以清除气泡的解决方案。 </li><li>我们小心地撒入模具混合，然后才开始巩固使用10毫升吸管。每个模具将举行约1毫升的粘稠液体。尽量避免在此过程中引入的气泡。 </li><li>小心地转移包含铸铁构造板块的孵化器。约30分钟后，从孵化器中取出的凝固结构和仔细添加成肌细胞培养基，每孔盖组织结构。返回板的孵化器（图2）。 </li></ol>第5部分：代表结果当正确执行，这个协议是在体内植入的成肌细胞中的组织构造（即从模具中删除时） 或进一步在体外分析，术后第2天。 <img src="/files/ftp_upload/1137/Figure1.jpg" alt="" /> 图1。完成兴建模具（见步骤1.5）。 <img src="/files/ftp_upload/1137/Figure2.jpg" alt="" width="400" height="321" /> 图2凝固成肌细胞中的组织构造[1] 。 <img src="/files/ftp_upload/1137/Figure3.jpg" alt="" /> 图3。一个成肌细胞含组织的H＆E染色切片构造。 “”描绘了一个纵向节和“B”显示了一个横截面。

<ol>
	<li>Choi, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+conduction+through+engineered+tissue.">Cardiac conduction through engineered tissue.</a> Am J Pathol. 169 (1), 72-85 (2006).</li><li>Rando, T. A., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+mouse+myoblast+purification,+characterization,+and+transplantation+for+cell-mediated+gene+therapy.">Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy.</a> J Cell Biol. 125 (6), 1275-1287 (1994).</li><li>Blau, H. M., Webster, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+human+muscle+cells.">Isolation and characterization of human muscle cells.</a> Proc Natl Acad Sci U S A. 78 (9), 5623-5627 (1981).</li><li>Powell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+human+bioartificial+muscles+expressing+a+foreign+recombinant+protein+for+gene+therapy.">Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy.</a> Hum Gene Ther. 10 (4), 565-577 (1999).</li><li>Vandenburgh, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+skeletal+muscle+organoids+for+reversible+gene+therapy.">Tissue-engineered skeletal muscle organoids for reversible gene therapy.</a> Hum Gene Ther. 7 (17), 2195-2200 (1996).</li><li>Gallop, P. M., Seifter, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+Properties+of+Soluble+Collagens.">Preparation and Properties of Soluble Collagens.</a> Methods in Enzymology. 6, 635-641 (1963).</li></ol>

机构动物护理和使用委员会在波士顿儿童医院的规定的准则和法规的规定进行了动物实验。

在其中的组织构造将铸模可在任何形状和大小，但需要有至少有两个附着点。否则，基质和细胞形成一个球形结构和细胞死亡。在本议定书中，我们描述了一个用于此目的的聚酯网的使用，但我们也成功地应用于不锈钢网。显然，规模较大的模具将需要更多的细胞和其他成分的体积较大。当模具，重要的是尽量减少使用硅酮胶量，以确保它是在位于油管非常两端。这是因为附近的胶粘剂的成肌细?...

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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>Silicone tubing</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>60985-724</td>
<td>&nbsp;</td>
<tr>
<td>Silicone adhesive</td>
<td>&nbsp;</td>
<td>Rhodia Silicones</td>
<td>MED ADH 4300 RTV</td>
<td>&nbsp;</td>
<tr>
<td>Polyester Mesh</td>
<td>&nbsp;</td>
<td>McMaster-Carr</td>
<td>93185T17</td>
<td>&nbsp;</td>
<tr>
<td>Laminin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L2020</td>
<td>&nbsp;</td>
<tr>
<td>Nutrient Mixture F-10 HAM</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>N6908</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Atlanta Biologicals</td>
<td>S11550</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Fungizone</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15290-018</td>
<td>&nbsp;</td>
<tr>
<td>Dispase-2</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>10295825001</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase 2</td>
<td>&nbsp;</td>
<td>Worthington</td>
<td>46H8863</td>
<td>&nbsp;</td>
<tr>
<td>Basic Fibroblast Growth Factor</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>G5071</td>
<td>&nbsp;</td>
<tr>
<td>150 mm tissue culture dishes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>353025</td>
<td>&nbsp;</td>
<tr>
<td>0.05% (1X) Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25300</td>
<td>&nbsp;</td>
<tr>
<td>1X Hanks Balanced Salt Solution</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14170-112</td>
<td>&nbsp;</td>
<tr>
<td>7.5% NaHCO3</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25080-094</td>
<td>&nbsp;</td>
<tr>
<td>70 &mu;m cell strainer</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>6-well plates</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>353046</td>
<td>&nbsp;</td>
<tr>
<td>50 mL Conical Vial</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352098</td>
<td>&nbsp;</td>
<tr>
<td>15 mL Conical Vial</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352099</td>
<td>&nbsp;</td>
<tr>
<td>0.2 &mu;m filter</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>194-2520</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

fabrication of myogenic engineered tissue constructs

尽管事实上，电子起搏器的寿命，节省了医疗设备，他们长期在儿科患者的表现可能会出现问题，由于孩子的小规模的和不可避免的增长所施加的限制。因此，真正需要的是一个专门设计的儿科患者心律紊乱的创新疗法的。我们提出一个含有胶原蛋白为基础的矩阵组成一个导电的生物替代autologously衍生细胞能更好地适应经济增长，减少经常性手术的需要，并大大提高这些患者的生活质量。在本研究中，我们描述了一种结合骨骼肌成肌细胞内的水凝胶矩阵的主要文化时尚手术植入的组织构造，将作为心脏的上下商会之间的电气管道的过程。最终，我们预计，使用这种类型的工程组织，以恢复完整的心脏传导阻滞的儿童房室导电。鉴于此，我们隔离新生儿Lewis大鼠和板到层粘连蛋白涂层的组织培养皿中，使用的既定协议的修改后的版本的骨骼肌成肌细胞<sup&gt; [2，3]</sup&gt;。一两天后，收集和混合培养的细胞用抗生素治疗，1型胶原，基质胶™，和碳酸氢钠<sub&gt; 3</sub&gt;。结果是一种粘稠的，统一的解决方案，可以投成几乎任何形状和大小的模具<sup“[1，4，5]</sup&gt;。对于我们的组织结构，我们聘请1型胶原蛋白的分离，从胎儿的羔羊皮肤使用的标准程序<sup[6]</sup&gt;。一旦组织已经凝固在37℃，培养基是经过精心添加到板块构造，直到被淹没。该工程的组织，然后是允许进一步凝聚通过2天以上脱水，此时它已准备就绪，<em&gt;在体外</em&gt;评估或手术植入。

Watch this Scientific Journal Video about Fabrication of Myogenic Engineered Tissue Constructs at JoVE.com

Fabrication of Myogenic Engineered Tissue Constructs

在这里，我们展示了制造胶原蛋白为基础，组织骨骼肌成肌细胞构造。这些工程结构的3 - D可用于更换或修复组织<em&gt;在体内</em&gt;。对于我们而言，我们设计的这些电气管道修复完整的心脏传导阻滞房室<sup“[1]</sup&gt;。

制作生肌工程的组织结构

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

fabrication-of-myogenic-engineered-tissue-constructs

Research

JoVE Journal

Biology

10.4K 次观看.  Children's Hospital Boston and Harvard Medical School. 在这里，我们展示了制造胶原蛋白为基础，组织骨骼肌成肌细胞构造。这些工程结构的3 - D可用于更换或修复组织在体内。对于我们而言，我们设计的这些电气管道修复完整的心脏传导阻滞房室

视频: Fabrication of Myogenic Engineered Tissue Constructs

PDMS Device Fabrication and Surface Modification

PDMS的设备制造和表面改性

科研

生物学

Fabrication of the Thermoplastic Microfluidic Channels

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using polymer/nanoparticle composite microscale lysis and solid phase extraction columns. The recovered samples are concentrated, small volume samples that are PCRable, without any additional cleanup. Here, we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then, we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns. We demonstrate grafting and polymerization of a carbon nanotube/polymer composite column for bacterial cell lysis. We then show the lysis process followed by solid phase extraction of nucleic acids from the sample on chip using a silica/polymer composite column. The attached protocols contain detailed instructions on how to make both lysis and solid phase extraction columns.

Here we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns.

制备热塑性微流体通道

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using ...

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we describe the techniques needed to generate low noise (&lt;0.5 pA) electrodes.  The techniques have been modified from published descriptions by previous researchers (1,2).  Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber.  Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip.  The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal.  Lastly, the electrodes are placed in an oven to cure the epoxy.  Careful handling of the electrodes is critical to ensure that they are made consistently and without damage.  This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

安培电极的制备

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we ...

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

大鼠心脏中植入的工程组织

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Mouse Sperm Cryopreservation and Recovery using the I&amp;middot;Cryo Kit

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

精子冷冻保存和恢复使用I ·冷冻包

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

重组工程同源重组构建<em&gt;果蝇</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

原发性生肌前体细胞/成肌细胞从基底哺乳动物支系文化准备

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.

针对性斯尔纳的噬菌体介导的交付构造击倒基因表达<em&gt;电子。大肠杆菌</em

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

在出生后的小鼠听泡解剖：中耳骨骼和组织学分析的分离

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...