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 ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Newt Lens Rejenerasyon Eğitim Kültürleme İris Pigment Epitel Hücreler için Sistem

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

Uygulamalarda Kantitatif Karşılaştırılması Cis</emTransgenik>-Regulatory Element (CRE) Etkinlikler Drosophila melanogaster</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

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.

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 ...

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, distinct microenvironments can impose differentiation, migration, and proliferation phenotypes, and on the tissue level the microenvironment processes as complex as morphogenesis and tumorigenesis1. Not only do the cell and molecular contents of microenvironments impact the cells within, but so do the elasticity2 and geometry3 of the tissue. Defined as the sum total of cell-cell, -ECM, and -soluble factor interactions, in addition to physical characteristics, the microenvironment is complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours.
Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments4. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes.
MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the availability of specific reagents. MEArrays are amenable to time-lapsed analysis, but most often are used for end point analyses of cellular functions that are measureable with fluorescent probes. For instance, DNA synthesis, apoptosis, acquisition of differentiated states, or production of specific gene products are commonly measured. Briefly, the basic flow of an MEArray experiment is to prepare slides coated with printing substrata and to prepare the master plate of proteins that are to be printed. Then the arrays are printed with a microarray robot, cells are allowed to attach, grow in culture, and then are chemically fixed upon reaching the experimental endpoint. Fluorescent or colorimetric assays, imaged with traditional microscopes or microarray scanners, are used to reveal relevant molecular and cellular phenotypes (Figure 1).

Fabrication and Use of MicroEnvironment microArrays MEArrays

A combinatorial functional screening method for gaining insights into the impacts of the molecular composition of microenvironments on cellular functions is described. The method takes advantage of existing microarray-based technologies to generate arrays of defined combinatorial microenvironments that support cell adhesion and functional analysis.

Fabrikasyon ve mikroçevresindeki microarrays Kullanımı (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, ...

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 ...

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