, 쓰래서 연구 기금에서 새 연구원 수상하고, 어린이 병원 보스턴에서 심장 전도 기금에 기부,이 작품은 국립 보건원에서 연구 기금 (HL088206 HL068915)에 의해 지원됩니다.

1 부 : 주조 금형을 구축 조립 <ol><li> 실리콘 튜브 (VWR)를 이등분하고 3cm 길이 조각으로 그것을 잘라 면도날을 사용합니다. </li><li> 튜브의 각 끝 안쪽에 이식 등급 RTV 실리콘 접착제 (Rhodia)의 한 방울을 넣으십시오. </li><li> 빨리 실리콘 접착제 드롭에 폴리에스터 메쉬의 작은 조각 (1cm X 1cm) (맥매스터 - 카)를 장소와 튜브의 끝 부분으로 맞춥니다. 이것은 건설 첨부 파일 약간 제기하고 평평한 표면을 제공합니다. 다른 쪽 끝을 위해 반복합니다. </li><li> 금형 3 일 동안 실온에서 건조하도록 허용합니다. 이들은 본질적으로 일회용 있으므로 한 번에 20-30 금형을 만들기 위해 도움이 그것입니다. </li><li> 접착제가 완전히 건조되면 그들이 필요 때까지, 70 % 에탄올로 가득 비커 이러한 금형을 저장합니다. 이 단계는 금형을 소독 도움이 될 것입니다 그리고 완성된 제품은 그림 1에 표시됩니다. </li></ol> 2 부 : myoblast 세포 격리를위한 준비 <ol><li> 250 1 MG Laminin (시그마)를 희석 ML 0.22 μm의 필터 - 멸균 인산 버퍼 호수 (PBS)가 1퍼센트 페니실린 / 스트렙토 마이신 (Invitrogen)과 1 %의 Fungizone (Invitrogen)를 포함. </li><li> 4 단계 1.1에서 희석 Laminin 솔루션의 ML과 기본 골격 myoblasts을 도금 전 최소한 4 시간 동안 37 º C에서 부화와 코트 150mm 조직 문화 플레이트 (BD 팔콘). </li><li> (20 % FBS (아틀란타 체액), 5 NG / L 기본 Fibroblast의 성장 인자 (Promega), 1 % 페니실린 / 스트렙토 마이신 (Invitrogen), 그리고 1 %의 Fungizone와 햄의 F - 10 영양소 혼합 (시그마)를 혼합하여 myoblast 미디어 만들기 Invitrogen). </li><li> 100 ML 2.4 U / ML Dispase - 2 (로체)에서 ~ 295 U / MG Collagenase 2 (워싱턴)의 1g을 해소하여 골격 근육 소화 솔루션을 준비합니다. 0.037 g CaCl 2를 추가하고 잘 섞는다. -20 ° C.에 10 ML 주사기 및 매장 aliquots에 장착되어 0.2 μm의 필터 디스크를 사용하여 소화 버퍼를 검사 필터 ° C myoblast 미디어와 함께 37 조직 소화에 필요한 금액을 넣으십시오. </li></ol> 파트 3 : 골격근에서 Myoblast 절연 <ol><li> 포셉 작은 가위를 사용하여, 척수를 따라 다시 아래로 깔끔히 피부와 근막의 레이어를 다시 필링 후 근육을 절개하여 죽은 신생아 루이스 쥐에서 paraspinal 근육을 제거합니다. </li><li> 조직 문화 후드에서 얼음 1X 행크스 밸런스드 소금 솔루션의 40 ML (HBSS) (Invitrogen)로 가득 50 ML 원뿔 튜브 (BD 팔콘)에 excised 근육을 놓으십시오. 근육 스트립​​은 튜브의 바닥에 정착 조심스럽게 문화 후드 (베이커)에 살균 파스퇴르 피펫과 진공 흡입하여 액체와 혈액 잔존물의 대부분을 제거할 수 있습니다. </li><li> 150mm 문화 요리에 수확 근육 조각을 붓고 및 흡입과 가능한 한 남은 액체의 많은를 제거합니다. 2 개의 싱글 - 양날 면도날을 사용하면 두꺼운 붙여 조직을 말하다. 붙여넣기 사전 예열 소화 솔루션을 붓고와 10 ML의 피펫을 사용하여 새로운 50 ML 튜브에 혼합을 전송하기만하면됩니다. 효소 소화하는 동안 조직은 때때로 피펫 - 에이드 (드루먼드) 무선에 설치되어 10 ML의 피펫과 (버블링 제외) triturated입니다. 다시 말하지만, 조직이 여분의 액체를 해결하고 제거할 수 있습니다. </li><li> 락을 플랫폼에서 튜브를 삽입하고 약 30 분 37 º C에서 소화. </li><li> 근육이 액화되면 600 XG (베크맨 GP) 10 분 실온에서 70 μm의 셀 스트레이너 (BD 팔콘)와 원심 분리기를 통해 솔루션을 전달합니다. 파스퇴르 피펫과 흡입으로 많은 액체가 가능한 제거하고 따뜻한 myoblast 매체 10 ML와 펠렛을 resuspend. </li><li> 사전 플레이트 15 분 uncoated 150mm 조직 문화 판에있는 세포는 섬유아 세포를 제거하는 데 도움. </li><li> 아직 laminin - 코팅 cultureware에 연결된 모든 1-2 쥐 새끼 한 접시가 너무 희석하지 않은 세포를 전송합니다. 37 ° C와 5 % CO 2에서 humidified 보육 세트에서 접시를 놓습니다. </li></ol> 4 부 : 주조 설계 조직 구조 (5 ML) <ol><li> 1-2일 후, 인큐베이터에서 150mm의 myoblast 조직 문화 접시 10을 제거 37 ° C 1X PBS로 두 번 그들을 씻어하고 각 플레이트에 따뜻한 0.05 % 트립신 - EDTA (에틸렌 다이아 민 테트라 초산) 4 ML를 추가합니다. 5 분 인큐베이터에 번호판을 반환합니다. </li><li> 문화 후드에있는 접시에서 분리 myoblasts을 수확하고 50 ML 튜브에 액체 (BD 팔콘)를 수집합니다. 접시를 모두 씻어 원심 튜브에 세포와 이것을 결합하는 myoblast 매체 10 ML을 사용합니다. 펠렛은 600 XG (베크맨 GP) 10 분 상온에서 세포 단계 3.5에서 설명한대로 초과하는 액체를 제거합니다. </li><li> 얼음 myoblast 매체와 장소의 1.6 ML에 myoblast 세포를 Resuspend. </li><li> 포셉를 사용하면, 신중하게 단계를 1.5에서 4 구조 금형을 제거하고 1X PBS로 두 번 그들을 씻어. 6 잘 조직 세제곱의 각 금형을 삽입하기 전에 파스퇴르 피펫과 함께 PBS의 모든 흔적에서 진공 흡입lture 플레이트 (BD 팔콘). </li><li> 얼음 구축 준비에 필요한 다른 시약을 (10X 햄의 F10, 항생제, 1을 입력 콜라겐 [3.9 MG / ML] [6]과 Matrigel ™) 모아서 37 º C.에 myoblast 미디어를 유지 500 μL 10X 햄의 F10, 100 μL 페니실린 / 스트렙토 마이신, 100 μL Fungizone, 1.6 ML의 콜라겐 및 15 ML 원뿔 관에 750 μL Matrigel ™를. 결합 10 초가 얼음위에 10로 설정 미니 Vortexer (VWR)과 장소를 사용하기위한 솔루션을 섞는다. </li><li> 다음 단계를 3.5에서 혼합 80 NaHCO 3 μL와 단계 3.3 myoblast 서스펜션을 추가합니다. 10 초에 대한 소용돌이 전체 혼합물과 거품의 솔루션을 취소하고 몇 분 얼음 양동이에 튜브를 반환합니다. </li><li> 그것이 10 ML의 피펫을 사용하여 응고하기 시작하기 전에 금형에 혼합물을 조심스럽게을 캐스팅. 각각의 금형은 점성 액체의 약 1 ML를 개최합니다. 이 절차를 수행하는 동안 공기 방울을 도입 피하기 위해보십시오. </li><li> 인큐베이터에 캐스트 구조가 들어있는 접시를 주의깊게 전송합니다. ~ 30 분 후 인큐베이터에서 확정 구성을 제거하고 조심스럽게 조직 구성 충당하기 위해 각 잘하는 myoblast 매체를 추가합니다. 인큐베이터 (그림 2)에 접시를 반환합니다. </li></ol> 제 5 부 : 대표 결과 이 프로토콜이 제대로 실행되면 myoblast 함유 조직 구조는 생체내 주입에 대한 준비가되어 (몰드에서 제거되면 IE) 또는 2 일 후 체외 분석에서 추가하십시오. <img src="/files/ftp_upload/1137/Figure1.jpg" alt="" /> 완료 금형 구조 그림 1. 예 (단계 1.5 참조). <img src="/files/ftp_upload/1137/Figure2.jpg" alt="" width="400" height="321" /> 그림 2. 확정 myoblast 함유 조직 [1]을 구축. <img src="/files/ftp_upload/1137/Figure3.jpg" alt="" /> 그림 3. myoblast 함유 조직의 H &amp; E 스테인드 섹션 구성. &quot;A&quot;는 세로 부분을 묘사하고 &quot;B&quot;는 단면을 보여줍니다.

<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>

동물 실험은 어린이 병원 보스톤에있는 기관 애니멀 케어 및 사용위원회에 의해 명시된 지침과 규정에 따라 수행되었다.

조직 구조가 캐스팅 될있는 금형은 어떤 모양과 크기로 만들 수 있지만, 첨부 파일이 최소한 두 지점이있을 필요합니다. 그렇지 않으면, 매트릭스 및 세포는 구형 구조를 형성하고 세포는 죽지. 현재 프로토콜에서, 우리는이 목적을 위해 폴리에스터 메쉬의 사용을 설명, 아직 우리는 성공적으로 스테인리스 메쉬를 사용했습니다. 물론, 큰 금형보다 세포와 다른 재료의 더 큰 볼륨을 요구​​할 것...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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

전자 맥박 조정기가있다는 사실에도 불구하고 생명을 구하는 의료 기기, 소아 환자에서 장기적인 성능이 아이의 작은 크기와 자신의 피할 수없는 성장에 의해 부과된 제한 문제 때문에 수 있습니다. 따라서 심장 리듬 장애와 소아 환자를 위해 특별히 설계된 혁신적인 치료법에 대한 진정한 필요가있다. 우리는 그 제안이 들어있는 콜라겐 기반 매트릭스로 구성된 전도성 생물 학적 대체 세포가 더 나은 성장에 적응 반복 수술의 필요성을 절감하며 크게 다음과 환자의 삶의 질을 향상시킬 수 autologously - 파생. 현재 연구에서 우리는 패션에 히드로겔 매트릭스 내에있는 마음의 상단과 하단 회의소 사이 전선관 될 것입니다 수술 - implantable 조직 구조를 기본 골격 myoblast 세포 문화를 통합을위한 절차를 설명합니다. 궁극적으로, 우리는 완전한 심장 블록과 어린이 방실 전기 전도를 복원할 수 있도록 설계 조직의이 유형을 사용하여 기대하고 있습니다. 그런 관점에서, 우리는 신생아 루이스 쥐 및 플레이트 그 규칙을 수정한 버전을 사용 laminin - 코팅 조직 문화 요리에의 골격 근육에서 myoblasts을 분리<sup&gt; [2, 3]</sup&gt;. 1~2일 후, 교양 세포는 항생제로 1을 입력 콜라겐, Matrigel ™, NaHCO를 수집하고 혼합<sub&gt; 3</sub&gt;. 그 결과 거의 모든 모양과 크기의 몰드로 주조 수있는 점성, 균일한 솔루션입니다<sup&gt; [1, 4, 5]</sup&gt;. 우리의 조직​​ 구조에 대한, 우리는 표준 절차를 사용하여 태아의 양고기 피부에서 분리된 제 1 형 콜라겐을 채용<sup&gt; [6]</sup&gt;. 일단 조직이 37 확정했다 ° 구조가 변할 때까지 C는, 문화, 미디어 조심스럽게 판에 추가됩니다. 설계 조직은 다음이 준비되는 시점에서 2 일간 더 탈수를 통해 더욱 응축 수 있습니다<em&gt; 체외에서</em&gt; 평가 또는 외과 - 이식.

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

Fabrication of Myogenic Engineered Tissue Constructs

여기, 우리는 콜라겐 기반 조직 골격 myoblasts를 포함하는 구성의 제조를 보여줍니다. 이러한 3 - D 설계 구조는 조직을 바꾸거나 수리하는 데 사용할 수 있습니다<em&gt; 생체내에</em&gt;. 우리 목적을 위해서도, 우리는 완전한 심장 블록의 수리를 위해 방실 전선관 이러한 디자인이<sup&gt; [1]</sup&gt;.

Myogenic 엔지니어링된 조직 구조의 제작

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 조회수.  Children's Hospital Boston and Harvard Medical School. 여기, 우리는 콜라겐 기반 조직 골격 myoblasts를 포함하는 구성의 제조를 보여줍니다. 이러한 3 - D 설계 구조는 조직을 바꾸거나 수리하는 데 사용할 수 있습니다 생체내에. 우리 목적을 위해서도, 우리는 완전한 심장 블록의 수리를 위해 방실 전선관 이러한 디자인이 [1].

비디오: Fabrication of Myogenic Engineered Tissue Constructs

PDMS Device Fabrication and Surface Modification

PDMS 장치 제작 및 표면 변경

연구

생물학

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.

열가 소성 Microfluidic 채널 제작

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.

굴지 공동 침강 분석, F - 굴지에 바인딩 단백질의 분석

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.

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.

랫 중심에 엔지니어링된 조직의 주입

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.

전 · Cryo 키트를 사용하여 마우스 정자 Cryopreservation 및 복구

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.

에서 상동 재조합 구문을 Recombineering<em&gt; 초파리</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.

기초 척추 계통의 주요 근원 성 전구 세포 / 근원 세포 배양의 준비

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.

표적 스르나의 파지 매개 배달에 유전자 발현을 허물고 구축<em&gt; E. 대장균</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.

출생 후의 쥐의 청각 수포의 해부 : 중이의 뼈와 조직 학적 분석의 분리

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 ...