Yeni Teşvik Ödülü, Thrasher Araştırma Fonu ve Boston Çocuk Hastanesi&#39;nden Kalp İletim Fonu katkı; Bu çalışma Ulusal Sağlık Enstitüsü&#39;nün araştırma hibeleri (HL088206 HL068915) tarafından desteklenmektedir.

Bölüm 1: döküm kalıpları inşa birleştirin. <ol><li> Yarıya silikon tüp (VWR) ve 3 cm uzunluğunda parçalar halinde kesmek için jilet kullanın. </li><li> Hortumun her iki ucundaki iç implant dereceli RTV silikon yapıştırıcı (Rhodia) bir damla yerleştirin. </li><li> Silikon yapıştırıcı açılan küçük bir parça polyester örgü (1 cm x 1 cm) (McMaster-Carr) hızlı bir şekilde yerleştirin ve hortumun ucu ile aynı hizaya getirin. Bu yapı eki için biraz yükseltilmiş ve düz bir yüzey sağlayacaktır. Diğer ucu için tekrarlayın. </li><li> Kalıp, 3 gün boyunca oda sıcaklığında kurumaya bırakın. Bu aslında tek olarak, bir defada 20 ila 30 kalıp yapmak için yararlı olur. </li><li> Yapıştırıcı tamamen kuruduktan sonra ihtiyaç duydukları kadar,% 70 etanol ile dolu bir beher bu kalıpları saklamak. Bu adım kalıpları sterilize yardımcı olacak ve bitmiş ürün Şekil 1&#39;de gösterilmiştir. </li></ol> Bölüm 2: myoblast hücre izolasyonu için Hazırlık <ol><li> 250 1 mg Laminin (Sigma) sulandırınız ml 0.22 mikron filtre ile sterilize edilmiş fosfat tamponlu salin (PBS) ve% 1 penisilin / Streptomisin (Invitrogen) ve% 1 Fungizone (Invitrogen) içeren. </li><li> Coat 150 mm 4 seyreltilmiş Laminin çözüm adımı 1.1 mL ve birincil iskelet matür kaplama önce en az 4 saat süreyle 37 º C&#39;de inkübe doku kültürü levhalar (BD Falcon). </li><li> (% 20 FBS (Atlanta Biyolojik), 5 ng / L temel Fibroblast Büyüme Faktörü (Promega), 1% Penisilin / Streptomisin (Invitrogen), ve% 1 Fungizone Ham F-10 Besin Karışım (Sigma) karıştırma myoblast orta olun Invitrogen). </li><li> 1 g, 100 mL 2.4 U / ml Dispase-2 (Roche) ~ 295 U / mg kollajenaz 2 (Worthington) eriterek iskelet kas sindirim çözüm hazırlayın. 0.037 g CaCl 2 ekleyin ve iyice karıştırın. -20 ° C&#39;de 10 ml şırınga ve mağaza alikotları takılan bir 0,2 mikron filtre disk kullanarak sindirim tampon sterilize filtreleme 37 ° C myoblast medya ile birlikte doku sindirim için gerekli olan miktar koyun. </li></ol> Bölüm 3: iskelet kas Myoblast izolasyonu <ol><li> Forseps ve küçük bir makas kullanarak, omurilik boyunca arka dilimleme cilt ve fasya tabakaları geri soyma ve sonra kasların eksize ölü yenidoğan Lewis sıçan paraspinal kaslar çıkarın. </li><li> Bir doku kültürü kaputu, buz üzerinde 1X Hanks Dengeli Tuz Çözeltisi 40 ml (HBSS) (Invitrogen) ile dolu bir 50 ml konik tüp (BD Falcon) eksize kasları yerleştirin. Kas şeritleri tüp altına yerleşmek ve dikkatli bir kültür kapağı (Baker), steril bir Pasteur pipet yardımıyla vakum emme sıvı ve kan kalıntılarının en çıkarmak için izin verin. </li><li> Hasat kas adet 150 mm kültür çanak üzerine dökün ve mümkün olduğunca emme ile kalan sıvı kadar kaldırın. 2 tek kenarlı Jilet kullanmak kalın bir macun doku kıyma. Hamurun üzerine dökün ve önceden ısıtılmış sindirim çözüm karışımı 10 ml pipet kullanarak yeni bir 50 ml tüp transferi. Enzimatik sindirim sırasında, doku bazen telsiz Pipet-Aid (Drummond) takılan 10 ml pipetle (köpüren olmadan) triturated konumundadır. Yine, aşırı sıvı doku yerleşmek ve kaldırmak için izin verir. </li><li> Sallanan bir platform üzerinde tüp yerleştirin ve yaklaşık 30 dakika süreyle 37 º C&#39;de sindirimi. </li><li> Kas sıvılaştırılmış sonra, 600 xg (Beckman GP) 10 dakika boyunca oda sıcaklığında 70 mikron hücre süzgecinden (BD Falcon) ve santrifüj yoluyla çözüm geçmek. Pasteur, pipet ve emme ile sıvı kadar mümkün olduğunca kaldırın ve tekrar süspansiyon, 10 ml sıcak myoblast orta pelet. </li><li> Ön plakası 15 dakika boyunca bir kaplanmamış 150 mm doku kültürü plaka hücreleri fibroblastlar kaldırmanıza yardımcı olmak için. </li><li> Henüz laminin kaplı Kültür varlıkları bağlı ve her 1 ila 2 sıçan yavrular için bir tabak olacak şekilde seyreltik hücreleri aktarın. 37 ° C ile% 5 CO 2 nemlendirilmiş bir inkübatör seti plaka koyun. </li></ol> Bölüm 4: Döküm mühendislik doku yapıları (5 ml) <ol><li> 1 ila 2 gün sonra, 10 inkübatör 150 mm myoblast doku kültürü tabak kaldırmak için, 37 ° C 1X PBS ile iki kez yıkayın ve sıcak,% 0.05 tripsin-EDTA 4 mL her plaka ekleyin. 5 dakika süreyle inkübatör plakaları geri dön. </li><li> Kültür kaputun içindeki plakalar müstakil matür Hasat ve 50 ml tüp içinde sıvı (BD Falcon) toplayabilir. 10 ml myoblast orta plakaların yıkayın ve santrifüj tüpüne hücreleri birleştirmek için kullanın. Pelet 600 xg (Beckman GP) 10 dakika oda sıcaklığında hücreleri ve adım 3.5 &#39;te açıklandığı gibi aşırı sıvı kaldırmak. </li><li> Buz üzerinde myoblast orta ve yerin 1.6 mL myoblast hücreleri tekrar </li><li> Forseps kullanarak dikkatli bir şekilde adım 1,5 &#39;dan 4 yapı kalıpları kaldırmak ve onları 1X PBS ile iki kez yıkayın. 6-iyi bir doku cu her kalıp yerleştirmeden önce Pasteur pipeti ile PBS tüm izlerini kapalı vakum emmelture plaka (BD Falcon). </li><li> Buz üzerinde yapı hazırlanması için gerekli diğer reaktifler (10X Ham F10, antibiyotikler, tip 1 kollajen [3,9 mg / ml] [6] ve Matrigel ™) toplayın ve 37 º C&#39;de myoblast medya tutmak 500 mcL 10X Ham F10, 100 mcL Penisilin / Streptomisin, 100 mcL Fungizone, 1.6 ml kollajen, ve 15 ml konik tüp içinde 750 mcL Matrigel ™ birleştirdik. 10 sn buz üzerinde 10 Mini-Vortexer (VWR) ve yeri için çözüm karıştırın. </li><li> Sonra, adım 3.5 80 NaHCO 3 mcL ve 3.3 adım myoblast süspansiyon karışıma ekleyin. 10 sn vorteksleyin tüm karışım ve baloncuklar çözüm temizlemek için birkaç dakika süreyle buz kovası tüp. </li><li> 10 ml pipet kullanarak katılaşmaya başlamadan önce kalıplar içine karışımı dikkatlice döküm. Her kalıp viskoz sıvı yaklaşık 1 ml yapacak. Bu işlem sırasında hava kabarcıkları tanıtan önlemek için deneyin. </li><li> Inkübatör dökme yapıları içeren plaka dikkatlice aktarın. ~ 30 dakika sonra, inkübatör katılaşmış yapıları kaldırmak ve doku inşa kapsayacak şekilde dikkatle, her bir kuyunun myoblast orta eklemek. Inkübatör (Şekil 2) plaka dönün. </li></ol> Bölüm 5: Temsilci sonuçları Bu protokol düzgün çalıştırıldığında, myoblast içeren doku inşa in vivo implantasyon için hazırdır (küf kaldırılır gibi) ya da 2 gün sonra in vitro analizler Daha fazla bilgi için . <img src="/files/ftp_upload/1137/Figure1.jpg" alt="" /> Şekil 1. tamamlanan yapı kalıp örnekleri (Adım 1.5 &#39;e bakınız). <img src="/files/ftp_upload/1137/Figure2.jpg" alt="" width="400" height="321" /> Şekil 2 katılaşmış myoblast içeren doku [1] inşa. <img src="/files/ftp_upload/1137/Figure3.jpg" alt="" /> Şekil 3: H &amp; E myoblast içeren doku lekeli bölümleri oluşturun . &quot;A&quot; uzunlamasına bir bölümü gösteriyor ve &quot;B&quot; bir kesit göstermektedir.

<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>

Boston Çocuk Hastanesi&#39;nden Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından belirlenen kurallara ve yönetmeliklere uygun olarak hayvanlar üzerinde deneyler yapıldı.

Olacağı doku inşa döküm kalıpları, herhangi bir şekil ve boyut olarak yapılabilir, ancak, ek, en az iki puan olması gerekir. Aksi takdirde, matriks ve hücrelerin küresel bir yapı oluştururlar ve hücreleri ölür. Bu protokolde, polyester mesh kullanımı bu amaç için bir tarif, ama biz de başarıyla kullanılan paslanmaz çelik hasır. Açıkçası, büyük kalıplar daha fazla hücre ve diğer malzemelerle daha büyük bir hacim gerektirir. Kalıpları yaparken, kullanılan silikon yapıştırıcı mi...

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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

Elektronik kalp pili olduğu gerçeğine rağmen yaşam tasarruf tıbbi cihazlar, pediatrik hastalarda uzun süreli performans bir çocuğun, küçük boyutları ve onların kaçınılmaz büyüme dayattığı kısıtlamaları sorunlu nedeniyle olabilir. Sonuç olarak, kalp ritmi bozuklukları olan pediatrik hastalar için özel olarak tasarlanmış yenilikçi tedaviler için gerçek bir ihtiyaç vardır. Önerdiğimiz içeren bir kolajen bazlı matris oluşan iletken bir biyolojik alternatif hücreler daha iyi, daha büyümeye uyum tekrarlayan ameliyatlar için ihtiyacını azaltır ve büyük ölçüde bu hastalar için yaşam kalitesini artırmak autologously-türetilmiştir. Bu çalışmada, biz moda hidrojel bir matriks içinde kalbin üst ve alt odacıkları arasındaki bir elektrik iletkeni olarak hizmet verecek bir cerrahi implant doku birincil iskelet myoblast hücre kültürleri birleştiren bir yapı için bir prosedür açıklanmaktadır. Sonuçta, biz, tam kalp bloğu olan çocuklar atriyoventriküler elektrik iletim geri yüklemek için bu tür mühendislik doku kullanarak tahmin ediyoruz. Bunun görünümünde matür, yenidoğan Lewis sıçan ve plaka kurulan protokolleri değiştirilmiş bir sürümünü kullanarak laminin kaplı doku kültürü yemekleri üzerine iskelet kasları izole<sup&gt; [2, 3]</sup&gt;. 1-2 gün sonra, kültür hücreleri, antibiyotik ile tip 1 kollajen, Matrigel ™, ve NaHCO toplanır ve karıştırılır<sub&gt; 3</sub&gt;. Sonucu hemen hemen her şekil ve büyüklükte bir kalıp içine dökme edilebilir bir viskoz, düzgün bir çözüm<sup&gt; [1, 4, 5]</sup&gt;. Doku yapıları, tip 1 kollajen standart prosedürler kullanarak fetal kuzu derisi izole istihdam<sup&gt; [6]</sup&gt;. Sonra doku, 37 katılaşmış ° C, yapı suya kadar kültür ortamı dikkatle plaka eklenir. Mühendislik doku daha sonra bunun için hazır olduğunu ve bu noktada, 2 gün daha dehidratasyon ile daha fazla yoğunlaşmaya izin verilir<em&gt; In vitro</em&gt; Değerlendirme veya cerrahi implantasyon.

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

Fabrication of Myogenic Engineered Tissue Constructs

Burada, kolajen doku, iskelet matür içeren yapıları imalatı göstermektedir. Bu 3-D mühendislik yapıları dokuların değiştirmek veya onarmak için kullanılan olabilir<em&gt; In vivo</em&gt;. Bizim amaçlarımız için, atriyoventriküler tam kalp bloğu tamiri için bir elektrik iletkeni olarak tasarladık<sup&gt; [1]</sup&gt;.

MİYOJEN Engineered Doku oluşturur imalatı

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 Görüntüleme.  Children's Hospital Boston and Harvard Medical School. Burada, kolajen doku, iskelet matür içeren yapıları imalatı göstermektedir. Bu 3-D mühendislik yapıları dokuların değiştirmek veya onarmak için kullanılan olabilir In vivo. Bizim amaçlarımız için, atriyoventriküler tam kalp bloğu tamiri için bir elektrik iletkeni olarak tasarladık [1].

Video: Fabrication of Myogenic Engineered Tissue Constructs

PDMS Device Fabrication and Surface Modification

PDMS Cihaz İmalat ve Yüzey Modifikasyonu

Biyoloji

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.

Termoplastik mikroakışkan Kanallar imalatı

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.

Aktin Co-Sedimantasyon Testi; F-Aktin Bağlayıcı Protein Analizi

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.

Amperometrik Elektrotlar imalatı

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.

Sıçan Kalp Engineered Doku implantasyonu

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.

I · Cryo Kit kullanarak Fare Sperm Kriyoprezervasyon ve Kurtarma

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.

Homolog rekombinasyon Oluşumlar Recombineering<em&gt; Drosophila</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.

Bazal Omurgalı soylarından İlköğretim andMyogenic Öncü Hücre / Miyoblast Kültürlerinin Hazırlanması

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.

Hedeflenen Srna faj aracılı teslim Gen İfade Knock Down için oluşturur<em&gt; E. E. coli</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.

Doğum sonrası Farelerde İşitsel Bulla diseksiyonu: Orta Kulak Kemikler ve histolojik analiz izolasyonu

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 ...

Methods Article

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