Name: dna electroporation, isolation and imaging of myofibers
Uploaded: 2015-12-23T15:00:00.000+00:00
Duration: 7 min 52 s
Description: Watch this Scientific Journal Video about DNA Electroporation, Isolation and Imaging of Myofibers at JoVE.com

이 작품은 NS047726, NS072027 및 AR052646은 건강의 국립 연구소에 의해 부여 지원되었다.

본 연구의 방법은 가이드 라인을 승인 의학 기관 동물 관리 및 사용위원회의 노스 웨스턴 대학 파인 버그 학교 (IACUC)와 윤리에 따라 수행되었다. 모든 노력이 고통을 최소화 하였다. 마우스의 굴근 심지 브레비스의 생체 내의 Electroporation (FDB) 근육 번들 1. 실험 절차 <ol><li> 포유 동물 세포에서 발현하는 것으로 알려져 프로모터를 사용하여 생체 내 발현을위한 디자인 플라스미드 (예를 들어, 거대 세포 바이러스, CMV) 또는 근육 (예를 들어, 근육 크레아틴 키나제, MCK) 6,7. 큰 플라스미드는 낮은 수준에서 표현 할 수 있습니다. CMV 프로모터 광범위 6,7 사용되어왔다. </li><li> E.로부터 플라스미드를 정화 대장균은 제조업체의 지침에 따라 내 독소 무료 조건을 사용하여. 5 μg의 플라스미드 / μL - 2에서 무균, 내 독소 무료 TE 버퍼에 플라스미드를 녹인다. </li><li> 희석 및 concentratio에 플라스미드 나누어지는멸균 독소 무료 TE (2 μg의 / μL) 10 μL에서 DNA의 20 μg의의 n은 멸균 microcentrifuge 관에 주입 당 버퍼. 주입 당 하나의 나누어지는을 준비합니다. </li><li> 8 단위의 최종 농도를 제공하는 칼슘 및 마그네슘없이 멸균 인산 완충 식염수에 1 배인 히알루로의 콘텐츠 - (- / PBS)에 희석한다. 나누어지는 멸균 microcentrifuge 관에 주입 당 희석 히알루 10 μL. </li><li> 진정 및 꼬리 또는 발가락 핀치에 응답 할 때까지 2.5 % 이소 플루 란을 사용하여 마우스를 마취. 정상 체온을 유지하기 위해 가열 패드 또는 가열 테이블에 앙와위에 동물을 놓는다. </li><li> 사전 기술 바늘 사이트 유래 바깥쪽으로 방사를 사용 포비돈 요오드 스크럽 알코올 교류 응용 3 회 마우스 발바닥을 청소한다. 이 기술은 병원균 도입을 최소화 무균 상태에서 바늘 위치를 유지한다. </li><li> 번째의 발바닥 주사피부와 멸균 1 ML의 주사기를 사용하여 근육 사이의 1 배의 히알루 용액 (2 ㎎ / ㎖ = 8 대)의 10 μL와 전자 마우스. 히알루는 근섬유로 플라스미드를보다 효율적으로 진입을 허용하는 세포 외 기질 성분의 소화. 발 뒤꿈치의 바닥에 입력하고 발가락쪽으로 바늘을 진행합니다. 바늘이 후진으로 천천히 액체를 놓습니다. 피부 아래에 주사 부위의 확장과 분홍색을 설정하기 시작합니다. </li><li> 반복 1.6 반대쪽 발에 1.7 단계를 반복합니다. </li><li> 마취를 분리하고 다시 새장에 마우스를 놓습니다. 마우스가 완전히 마취에서 복구 할 수 있습니다. </li><li> 2 시간을 기다립니다. </li><li> , 마취 및 발 소독 절차를 반복 1.5 및 1.6 단계를 반복합니다. </li><li> 히알루로니다 제와 같은 방식으로 발바닥 내로 희석 플라스미드 DNA를 주입. 최대 볼륨 20 μL 이하인지 확인합니다. 이중 발현 플라스미드 20의 최대 음량 각 플라스미드 20 μg의 주입# 181; L 이하이다. </li><li> 반대쪽 발바닥에 플라스미드 주입을 반복하거나 제어 주사에 대한 반대측 발을 사용합니다. </li><li> 처음에 주입 한 발에서 떨어져 미세 집게로 밑줄 근육으로부터 피부를 들어 올려 다리의 치유 발가락 라인에 수직 위치에 발가락 근처 피부의 공을 통해 하나 27 G 바늘을 놓습니다. 두 번째 멸균 27 G 바늘을 가지고 발 뒤꿈치에서 피부를 통해 수평으로 배치합니다. 장소 바늘 떨어져 각 ~ 다른 1cm 평행. </li><li> 바늘이 서로 접촉하지 않도록하고 병렬 바늘에 악어 클램프, 클램프 전극을 사용. 점토의 사용 장소에 클램프를 확보 할 수있다. </li><li> 전기 자극기에 전극을 연결합니다. </li><li> 100 V / cm에서, 1 Hz에서 20 펄스 지속 시간이 20 밀리 초 / 각을인가함으로써 근육 Electroporate. 발가락 자극하여 경련 수 있습니다. </li><li> 필요한 경우 반대쪽 발에 자극 절차를 반복합니다. </li><li>바늘을 제거합니다. 포비돈 요오드 스크럽과 발바닥을 닦습니다. </li><li> 마취를 끕니다. 복구 케이지와 모니터 활동에 마우스를 돌려줍니다. 예상대로 절차가 수행 된 경우, 동물을 30 분 내에 정상 보행을 회복 및 사전 주입 동작과 보행 또는 활성 수준에 차이를 나타내지 않는다한다. 따라서, 시술 후 진통제는 필요하지 않습니다. 복구시, 동식물 사육장에 동물을 반환합니다. 참고 : 단백질 발현은 전기 후 빠르면 48 시간을 분석 할 수 있습니다. 14 일 전기 (10) 후 - 그러나, 전기 후 더 완벽한 복구를 할 수 있도록, 우리는 7 고립과 근섬유의 연구를 선호한다. 식 4 주 후 일렉트로만큼 분석 될 수있다. </li></ol> 굴근 심지 브레비스 (FDB) 섬유의 분리 2. 실험 절차 <ol><li> 해동 콜라게나. 각 FDB 번들 12 웰 조직 배양 플레이트에 웰 두 준비. 하나에서 잘 1m를 추가미리 예열 둘 베코의 수정 이글스 미디어 플러스 소 혈청 알부민 (DMEM + BSA)의와 다른 난 잘 미리 예열 링거 액 1ml를 추가합니다. 또한, 요리를 실 가드 35mm에 1 배 링거 용액의 신선한 10ml를 추가합니다. </li><li> 죽음을 보장하기 위해 자궁 경부 전위 또는 다른 방법으로 다음, CO 2 마취 가스의 흡입을 통해 동물을 희생. </li><li> 깨끗한 면도날을 사용하여 발목 관절 위의 뒷다리를 제거하고 접시를 실 가드 35mm의 1 배 링거 용액에 잠수함. </li><li> 한 발 꽉 핀, 5 발가락 끝의 각 통해 발목에서 30 G 바늘과 실 가드 접시에, 발바닥이 위로 향하게. </li><li> 발목에서 시작, 조심하지 닉, 바로 발가락쪽으로 헤어 라인을 따라 피트의 측면을 피부 아래의 근육을 피부를 싹둑. 다리의 반대편에 반복합니다. </li><li> 미세 집게로 발목의 기지에서 피부를 잡고 발 뒤꿈치에서 피부를 잘라. </li><li> 피부를 리프팅피부로하지 닉 근육으로 가위를 가리, 집게와 플랩. 효과적으로 피부를 제거한 하부의 결합 조직이 잘린. </li><li> FDB 근육 다발이 뼈의 힘줄을 확보하기 위해 발 뒤꿈치에있는 큰 힘줄을 잘라 제거하십시오. 집게와 힘줄을 잡고 조심스럽게 가위로 FDB에 연결된 모든 결합 조직을 제거하는 큰 흰색 건에서 FDB 번들을 해부하다. 제대로하면 번들은 쉽게 개인의 자리로 이어지는 밝은 흰색 힘줄을 드러내는 기본 힘줄의 들어 올려됩니다. 발에서 번들을 확보 발가락 근처의 힘줄에서 FDB를 잘라. </li><li> 잘 포함하는 DMEM + BSA의 전체 근육 번들을 놓습니다. </li><li> 반대쪽 발에 반복합니다. </li><li> 일단 모든 FDB 근육은 각 웰에 포함 된 DMEM + BSA와 근육에 콜라겐의 100 μl를 추가, 격리됩니다. </li><li> 소화 이전에 거꾸로 형광 현미경에 전기의 성공을 확인합니다. 수행 properl 경우Y, 90 %의 형질 전환 효율은 위쪽으로 달성 될 수있다. </li><li> 약 1 시간 동안 10 %의 CO 2에 가습, 37 ° C를 인큐베이터에서 접시를 품어. 배양 시간은 질병 모델과 배경과 콜라겐의 많은에 따라 달라질 수 있습니다. </li><li> 1 시간 후, 조심스럽게 잘 포함 만 링거 용액에 FDB 번들을 전송합니다. </li><li> 1 ml의 피펫을 사용하여 링거 용액 웰 FDB 번들 씹다. 번들 쉽게 피펫 팁을 통과 할 수 있도록 깨끗한​​ 면도날 피펫 팁의 끝을 잘라. 조심스럽게 번들가 통과 팁과 평행 다운 할 수 있도록 근육 (~ 15 배)를 씹다. 본래 섬유는 링거 용액에 떨어질 것입니다. 섬유가 쉽게 번들을 떨어지지 않는 경우, 콜라겐 용액에 다시 근육을 놓습니다. </li><li> 판 당 접시 ~ 500 μL에 고립 된 섬유 플레이트. </li><li> 근육은 15 분 동안 잘에서 소화 할 수 있습니다. </li><li> 반복 2.15 단계2.17로 다발의 크기가 감소 될 때까지 섬유의 대부분은 번들 (통상 2-3 회 전체)로부터 해리된다. 일단 근육 용이 1 ml의 피펫 팁을 통과 해리. </li><li> 섬유가 15 분 이상 동안 접시에 부착하자. 신선한 링거 용액 시간 제약에 따라 잔여게나 희석 플레이트에 추가되거나 변경 될 수있다. 참고 : 섬유 이제 이미징을위한 준비가되어 있습니다. </li></ol> 공 초점 현미경을 사용하여 레이저 유도 멤브레인 손상의 시각화 3. 실험 절차 <ol><li> 로드 섬유 2.5 μm의 300 μL 링거 용액 FM 4-64 염료 FM 1-43으로 이미징 직전. 일반적으로, 분리 후 이미지 섬유 15 분까지 2 시간. 그러나, 섬유는 24 시간 후 분리까지 영상화 할 수있다. </li><li> UV 레이저 및 60X / 63X 대물 렌즈가 장착 된 공 초점 현미경에 유리 바닥 접시를 놓습니다. </li><li> 섬유를 찾습니다. 섬유가 단단히 연결되어 있는지 확인합니다현미경 기반을 눌러 접시에. 해리 과정에서 손상되었다 섬유를 제외합니다. 손상된 섬유 컬 막 소포, FM 염료의 흡수 높은 수준을 포함​​하거나 강렬하게 구부리 수 있습니다. </li><li> 섬유 막에 손상을 유도하는, 핵의 클리어 필드 촬상 창의 중심에 위치 sarcolemma. sarcolemma 매우 날카로운되도록 FM 염료 채널을 사용 sarcolemma에 초점을 맞 춥니 다. </li><li> FM 4-64 채널을 활용 sarcolemma에 투자 수익 (ROI) 크로스 헤어를 놓습니다. </li><li> 3 초 (~ X 350 nm의 영역 350 ㎚) 80 %의 전력에서 1 픽셀 지점을 조사. FM 염료는 부상의 사이트에서 셀을 입력합니다. 전력 및 시간을 증가 시키거나 모독의 영역을 감소하도록 조정될 수있다. </li><li> 손상시 손상하기 전에 매 2 초 후 손상, 최대 5 분 후 매 10 초 섬유의 이미지를 캡처합니다. 타이밍은 필요에 따라 조정될 수있다. 개별 이미지는 이미지 J.에서 시간 경과 영화로 변환 할 수 있습니다 </li><li> 단계를 반복합니다접시 당 최대 3 섬유에 3.7을 통해 3.3. </li></ol>

<ol>
	<li>Blau, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+the+differentiated+state.">Plasticity of the differentiated state.</a> Science. 230, 758-766 (1985).</li><li>Yaffe, D., Saxel, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+passaging+and+differentiation+of+myogenic+cells+isolated+from+dystrophic+mouse+muscle.">Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle.</a> Nature. 270, 725-727 (1977).</li><li>Richler, C., Yaffe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vitro+cultivation+and+differentiation+capacities+of+myogenic+cell+lines.">The in vitro cultivation and differentiation capacities of myogenic cell lines.</a> Dev Biol. 23, 1-22 (1970).</li><li>Yaffe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retention+of+differentiation+potentialities+during+prolonged+cultivation+of+myogenic+cells.">Retention of differentiation potentialities during prolonged cultivation of myogenic cells.</a> Proc Natl Acad Sci USA. 61, 477-483 (1968).</li><li>Mamchaoui, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalized+pathological+human+myoblasts:+towards+a+universal+tool+for+the+study+of+neuromuscular+disorders.">Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders.</a> Skelet muscle. 1, 34 (2011).</li><li>DiFranco, M., Quinonez, M., Capote, J., Vergara, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+transfection+of+mammalian+skeletal+muscles+using+in+vivo+electroporation.">DNA transfection of mammalian skeletal muscles using in vivo electroporation.</a> J Vis Exp: JoVE. , (2009).</li><li>Swaggart, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annexin+A6+modifies+muscular+dystrophy+by+mediating+sarcolemmal+repair.">Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair.</a> Proc Natl Acad Sci USA. 111, 6004-6009 (2014).</li><li>Kerr, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysferlin+stabilizes+stress-induced+Ca2++signaling+in+the+transverse+tubule+membrane.">Dysferlin stabilizes stress-induced Ca2+ signaling in the transverse tubule membrane.</a> Proc Natl Acad Sci USA. 110, 20831-20836 (2013).</li><li>Anderson, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+micropeptide+encoded+by+a+putative+long+noncoding+RNA+regulates+muscle+performance.">A micropeptide encoded by a putative long noncoding RNA regulates muscle performance.</a> Cell. 160, 595-606 (2015).</li><li>Roche, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+and+histological+changes+in+skeletal+muscle+following+in+vivo+gene+transfer+by+electroporation.">Physiological and histological changes in skeletal muscle following in vivo gene transfer by electroporation.</a> Am J Physiol Cell Physiol. 301, C1239-1250 (2011).</li><li>Reddy, A., Caler, E. V., Andrews, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membrane+repair+is+mediated+by+Ca(2+)-regulated+exocytosis+of+lysosomes.">Plasma membrane repair is mediated by Ca(2+)-regulated exocytosis of lysosomes.</a> Cell. 106, 157-169 (2001).</li><li>Wang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+blebbing+as+an+assessment+of+functional+rescue+of+dysferlin-deficient+human+myotubes+via+nonsense+suppression.">Membrane blebbing as an assessment of functional rescue of dysferlin-deficient human myotubes via nonsense suppression.</a> J Appl physiol. 109, 901-905 (2010).</li><li>Cai, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MG53+nucleates+assembly+of+cell+membrane+repair+machinery.">MG53 nucleates assembly of cell membrane repair machinery.</a> Nat Cell Biol. 11, 56-64 (2009).</li><li>Marg, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sarcolemmal+repair+is+a+slow+process+and+includes+EHD2.">Sarcolemmal repair is a slow process and includes EHD2.</a> Traffic. 13, 1286-1294 (2012).</li><li>Bansal, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+membrane+repair+in+dysferlin-deficient+muscular+dystrophy.">Defective membrane repair in dysferlin-deficient muscular dystrophy.</a> Nature. 423, 168-172 (2003).</li><li>Defour, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysferlin+regulates+cell+membrane+repair+by+facilitating+injury-triggered+acid+sphingomyelinase+secretion.">Dysferlin regulates cell membrane repair by facilitating injury-triggered acid sphingomyelinase secretion.</a> Cell Death Dis. 5, e1306 (2014).</li></ol>

관심의 충돌이 없습니다.

생체 내 형광 태그로 단백질을 연구하는 전기의 사용은 이상적으로 충실히 근육 전체 셀 구조를 복제 적합한 세포주 모델의 부재 주어진 근육의 연구를 위해 적합하다. 이 프로토콜은 레이저 상처 후 단백질 지역화 및 전좌의 상세한 화상을 제공하는 일렉트로 고해상도 공 초점 현미경의 사용률을 설명한다. 이 방법은 세포 골격 재구성, 인신 매매 및 융합을 포함한 다른 세포 과정을 연구?...

각각의 근섬유가 큰, 매우 조직 세포 융합 세포이다. 근육에 대한 몇 가지 세포 배양 모델이 있습니다; 그러나,이 모델은 완전히 성숙한 근섬유로 분화하지 않는 전공 제한으로있다. 예를 들어, L6 및 C2C12 세포주는 마우스 및 래트 각각 1-4로부터 유도된다. 혈청 기아 조건 하에서, 단핵 &quot;근육 모세포 같은&quot;세포 증식을 중단 세포주기 회수를 받아야하고 sarcomeres과 다핵 세포를 형성하는 근육 조직 프로그램에 입력 myotubes라고 함. 장기간 배양 조건으로, myotubes는 수축 특성을 나타낼 수 있으며, 문화의 &quot;트&quot;. 인간 세포주는 이제 5 확립되었다. 이들 불멸화 세포주 이외에, 단핵구는 근육 아세포로부터 myotubes을 형성 할 것이다 혈청 기아 유사한 조건 하에서 분리 될 수있다. 이들 세포주 및 일차 근원 세포 배양 물은 높은 사용 아르FUL들은 플라스미드로 형질 감염 또는 바이러스 형질 도입 및 염기성 세포 생물학적 과정을 연구하기 위해 이용 될 수 있기 때문이다. myotubes를 형성하도록 유도 된 경우에는, 이들 세포는 심지어, 성숙한 근육 조직의 많은 두드러진 특징이 부족하다. 구체적 myotubes 개별 성숙한 근섬유보다 훨씬 작고 근섬유의 정상적인 형상이 부족하다. 비판적으로, myotubes는 가로 (T-) 세관 부족, myoplasm에 걸쳐 효율적으로 칼슘 2 + 릴리스에 필요한 멤브레인 네트워크. 차 근원 세포 또는 근육 조직 세포 라인에 다른 방법은 성숙한 근섬유를 사용하여 수반한다. 형질 전환은 태그 된 단백질의 발현을 확립하기 위해 사용될 수 있지만,이 방법은 비용과 시간이 소요된다. 마우스 근육의 생체 전기는 속도 및 신뢰성 6-10위한 바람직한 방법으로서 떠오르고있다. 생체 전기와 근섬유 하를 효율적으로 분리하는 방법마우스 굴근 심지 브레비스 (FDB) 근육 (6)에 최적화되어했습니다. 방법은 쉽게 완료 플라스미드에서 생체 내 발현 유도하는 최소 침습 있습니다 할 수 있습니다. 이 방법은 현재의 sarcolemma -6,7- 레이저 파쇄 촬상 비롯한 고해상도 영상화 방법과 결합된다. 형광 염료 및 형광 표지 된 단백질의 발현이 결합 성숙한 근섬유 세포에서 생물학적 과정을 모니터링하는데 사용될 수있다.

<table><tbody><tr><td>DMEM</td><td>Life Technologies</td><td>11995-073</td><td>Dissection Media&lt;br/&gt; Dilution: 50 ml +100 g BSA</td></tr><tr><td>Collagenase, Type II</td><td>Life Technologies</td><td>17101-015</td><td>Fiber Digestion&lt;br/&gt; Dilution: 160 mg / 4 ml DMEM (stock 40 mg/ml aliquot)</td></tr><tr><td>Falcon 12-well dishes</td><td>Fisher Scientific</td><td>877229</td><td>Fiber Digestion</td></tr><tr><td>Ringers Solution</td><td></td><td></td><td>Fiber Digestion and Imaging&lt;br/&gt; Dilution: 146 mM NaCl&lt;br/&gt; 5 mM&amp;nbsp;&amp;nbsp; KCl&lt;br/&gt; 2 mM&amp;nbsp;&amp;nbsp; CaCl2&lt;br/&gt; 1 mM&amp;nbsp;&amp;nbsp; MCl2&lt;br/&gt; 10 mM HEPES&lt;br/&gt; pH7.4 in H2O</td></tr><tr><td>1 ml Pipettes</td><td>Axygen</td><td>T-1000-C-R</td><td>Digestion</td></tr><tr><td>Razor Blades</td><td>Personna</td><td>94-120-71</td><td>Digestion</td></tr><tr><td>Precision Glide 30 G needle</td><td>BD Biosciences</td><td>305128</td><td>Dissection</td></tr><tr><td>1x PBS (-/-)</td><td>Life Technologies</td><td>14190-250</td><td>Dissection</td></tr><tr><td>Sylgard 184</td><td>Fisher Scientific</td><td>50-366-794</td><td>Dissection</td></tr><tr><td>Forceps</td><td>FST by Dumont</td><td>#5/45</td><td>Dissection</td></tr><tr><td>Forceps</td><td>Roboz</td><td>RS-4913</td><td>Dissection</td></tr><tr><td>Scissors</td><td>World Precision Instruments</td><td>500260</td><td>Dissection</td></tr><tr><td>BSA</td><td>Sigma</td><td>A7906</td><td>Dissection media&lt;br/&gt; Dilution: 100 mg/50 ml DMEM</td></tr><tr><td>Falcon 60 mm dishes</td><td>Fisher Scientific</td><td>08772B</td><td>Dissection- used to hold sylgard</td></tr><tr><td>Clay</td><td></td><td></td><td>Electroporation</td></tr><tr><td>Stimulator&amp;nbsp;</td><td>Grass</td><td>s88x</td><td>Electroporation</td></tr><tr><td>Clamps</td><td>Radio Shack</td><td>270-356</td><td>Electroporation</td></tr><tr><td>Triadine</td><td>Aplicare</td><td>82-220</td><td>Electroporation</td></tr><tr><td>Precision Glide 27 G needle</td><td>BD Biosciences</td><td>305136</td><td>Electroporation</td></tr><tr><td>Simulator</td><td>Grass</td><td>SIU-V</td><td>Electroporation</td></tr><tr><td>Gauze</td><td>Covidien</td><td>2146</td><td>Electroporation</td></tr><tr><td>Hyaluronidase</td><td>Sigma</td><td>H4272</td><td>Injection&lt;br/&gt; Dilution: Add 160 ul PBS (1x) to 40 &amp;micro;l 5x stock</td></tr><tr><td>1 cc U-100 Insulin Syringe (28G1/2)&amp;nbsp;</td><td>BD Biosciences</td><td>329420</td><td>Injection</td></tr><tr><td>Eppendorf Tubes</td><td>Fisher Scientific</td><td>02-682-550</td><td>Injection</td></tr><tr><td>35 mm glass bottom Microwell dish</td><td>MaTek</td><td>P35G-1.5-14-C</td><td>Microscopy</td></tr><tr><td>FM 4-64</td><td>Life Technologies</td><td>T-13320</td><td>Microscopy&lt;br/&gt; Dilution: Final 2.5 ug/ml</td></tr><tr><td>FM 1-43</td><td>Life Technologies</td><td>T-35356</td><td>Microscopy&lt;br/&gt; Dilution: Final 2.5 ug/ml</td></tr><tr><td>Endotoxinfree Plasmid Maxi Kit</td><td>Qiagen</td><td>12362</td><td>Plasmid Purification</td></tr></tbody></table>

dna electroporation, isolation and imaging of myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

전기 효율적으로 굴근 심지 브레비스 (FDB) 근육 번들 (그림 1A)로 정제 된 플라스미드 DNA를 소개하는 최소 침습 기술이다. 이레는 형질 전환 형광 태그 단백질은 고립 된 근섬유 (그림 1B)에 가시화 게시 할 수 있습니다. 고립 된 섬유는 도금 공 초점 현미경 이미징에 대 한 준비가되어 있습니다. 섬유 레이저 유도 부상으로 실시 할 수있다. FM 염료 막...

Watch this Scientific Journal Video about DNA Electroporation, Isolation and Imaging of Myofibers at JoVE.com

DNA Electroporation, Isolation and Imaging of Myofibers

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

DNA 일렉트로, 근섬유의 분리 및 이미징

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

dna-electroporation-isolation-and-imaging-of-myofibers

Research

JoVE Journal

Biology

12.2K 조회수.  Northwestern University. This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure. 

비디오: DNA Electroporation, Isolation and Imaging of Myofibers

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Several mouse models have been created but they either do not truly represent the human physiopathology of the disease or are not representative of the broad spectrum of mutations found in humans. The immortalization of human primary myoblasts is an alternative to this limitation; however, it is still dependent on muscle biopsies, which are invasive and not easily available. In contrast, skin biopsies are easier to obtain and less invasive to patients. Fibroblasts derived from skin biopsies can be immortalized and transdifferentiated into myoblasts, providing a source of cells with excellent myogenic potential. Here, we describe a fast and direct reprogramming method of fibroblast into a myogenic lineage. Fibroblasts are transduced with two lentiviruses: hTERT to immortalize the primary culture and a tet-inducible MYOD, which upon the addition of doxycycline, induces the conversion of fibroblasts into myoblasts and then mature myotubes, which express late differentiation markers. This quick transdifferentiation protocol represents a powerful tool to investigate pathological mechanisms and to investigate innovative gene-based or pharmacological biotherapies for neuromuscular disorders.

This protocol describes the conversion of skin fibroblasts into myoblasts and their differentiation into myotubes. The cell lines are derived from patients with neuromuscular disorders and can be used to investigate pathological mechanisms and to test therapeutic strategies.

신경 근육 장애에 대 한 치료를 조사 하기 위해 Myoblasts에 인간 섬유 아 세포의 직접 재프로그래밍

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of ...

연구

유전학

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

사용 포유류 골격 근육의 유전자 Transfection VIVO에서 Electroporation

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

생물학

Minimally Invasive Muscle Embedding (MIME) - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new muscle fibers under the right conditions. Although SCs can be harvested from muscle tissue and cultured in vitro, the resulting myoblast cells are not very effective in promoting myogenesis when transplanted into host muscle. Surgically exposing the host muscle and grafting segments of donor muscle tissue, or the isolated muscle fibers with their SCs onto host muscle, promotes better myogenesis compared to myoblast transplantation. We have developed a novel technique that we call Minimally Invasive Muscle Embedding (MIME). MIME involves passing a surgical needle through the host muscle, drawing a piece of donor muscle tissue through the needle track, and then leaving the donor tissue embedded in the host muscle so that it may act as a source of SCs for the host muscle. Here we describe in detail the steps involved in performing MIME in an immunodeficient mouse model that expresses a green fluorescent protein (GFP) in all of its cells. Immunodeficiency in the host mouse reduces the risk of immune rejection of the donor tissue, and GFP expression enables easy identification of the host muscle fibers (GFP+) and donor-cell-derived muscle fibers (GFP-). Our pilot data suggest that MIME can be used to implant an extensor digitorum longus (EDL) muscle from a donor mouse into the tibialis anterior (TA) muscle of a host mouse. Our data also suggest that when a myotoxin (barium chloride, BaCl2) is injected into the host muscle after MIME, there is evidence of donor-cell-derived myogenesis in the host muscle, with approximately 5%, 26%, 26% and 43% of the fibers in a single host TA muscle showing no host contribution, minimal host contribution, moderate host contribution, and maximal host contribution, respectively.

Minimally Invasive Muscle Embedding MIME - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

We describe a novel experimental technique that we call Minimally Invasive Muscle Embedding (MIME), which is based on the evidence that skeletal muscle tissue contains viable myogenic cells that can facilitate donor-cell-mediated myogenesis when implanted into a host muscle.

최소한 침략 적 근육 (MIME) 포함-기증자 세포 중재 하는 Myogenesis를 촉진 하기 위하여 새로운 실험 기법

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new ...

의학

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

성인 골격근의 개인 Myofibers 및 위성 세포의 분리와 문화

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

해리 된 준비에서 배아와 애벌레 Zebrafish의 골격 근섬유의 분석

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

초기 산후 발달에 있는 에머리 드레이푸스 근 이영양증의 Murine 모형에서 단 근섬유 격리 및 문화

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear positioning occurs during skeletal muscle differentiation. Muscle fibers (myofibers) are multinucleated cells formed by the fusion of muscle precursor cells (myoblasts) derived from muscle stem cells (satellite cells) that undergo proliferation and differentiation. Correct nuclear positioning within myofibers is required for the proper muscle regeneration and function. The common procedure to assess myoblast differentiation and myofiber formation relies on fixed cells analyzed by immunofluorescence, which impedes the study of nuclear movement and cell behavior over time. Here, we describe a method for the analysis of myoblast differentiation and myofiber formation by live cell imaging. We provide a software for automated nuclear tracking to obtain a high-throughput quantitative characterization of nuclear dynamics and myoblast behavior (i.e., the trajectory) during differentiation and fusion.

Skeletal muscle differentiation is a highly dynamic process, which particularly relies on nuclear positioning. Here, we describe a method to track nuclei movements by live cell imaging during myoblast differentiation and myotube formation and to perform a quantitative characterization of nuclei dynamics by extracting information from automatic tracking.

악용 Myoblast 분화와 융합 하는 동안 트랙 핵에 라이브 영상

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear ...

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

골격 근육 생검에서 Myofibrils를 분리하고 나노 뉴턴 해상도 포스 트랜스듀서로 수축 기능을 결정합니다.

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

성인 근육 줄기 세포 기능 전 비보의 평가를위한 단일 근섬유 배양 분석

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

다양한 길이의 마우스 근육 섬유의 효소 분리 및 특성화

Intact Short, Intermediate, and Long Skeletal Muscle Fibers Obtained by Enzymatic Dissociation of Six Hindlimb Muscles of Mice: Beyond Flexor Digitorum Brevis

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal with the short fibers of the flexor digitorum brevis (FDB), which restrains the scope of results dealing with fiber types, limits the amount of biological material available, and impedes a clear connection between cellular physiological phenomena and previous biochemical and dynamical knowledge obtained in other muscles.
This paper describes how to obtain intact fibers from six muscles with different fiber type profiles and lengths. Using C57BL/6 adult mice, we show the muscle dissection and fiber isolation protocol and demonstrate the suitability of the fibers for Ca2+ transient studies and their morphometric characterization. The fiber type composition of the muscles is also presented. When dissociated, all muscles rendered intact, living fibers that contract briskly for more than 24 h. FDB gave short (&#60;1 mm), peroneus digiti quarti (PDQA) and peroneus longus (PL) gave intermediate (1-3 mm), while extensor digitorum longus (EDL), extensor hallucis longus (EHL), and soleus muscles released long (3-6 mm) fibers.
When recorded with the fast dye Mag-Fluo-4, Ca2+ transients of PDQA, PL, and EHL fibers showed the fast, narrow kinetics reminiscent of the morphology type II (MT-II), known to correspond to type IIX and IIB fibers. This is consistent with the fact that these muscles have over 90% of type II fibers compared with FDB (~80%) and soleus (~65%). Moving beyond FDB, we demonstrate for the first time the dissociation of several muscles, which render fibers spanning a range of lengths between 1 and 6 mm. These fibers are viable and give fast Ca2+ transients, indicating that the MT-II can be generalized to IIX and IIB fast fibers, regardless of their muscle source. These results increase the availability of models for mature skeletal muscle studies.

저자 스포트라이트: Isolation of Long Muscle Fibers from Mouse Hindlimb Muscles for Studying Excitation-Contraction Coupling Across Fiber Across Excitation-Contraction Coupling (섬유 유형 간 흥분-수축 결합 연구를 위한 쥐 뒷다리 근

We describe a protocol to obtain enzymatically dissociated fibers of different lengths and types from six muscles of adult mice: three of them already described (flexor digitorum brevis, extensor digitorum longus, soleus) and three of them successfully dissociated for the first time (extensor hallucis longus, peroneus longus, peroneus digiti quarti).

생쥐의 6개 뒷다리 근육의 효소 해리로 얻은 온전한 짧은, 중간 및 긴 골격근 섬유: Beyond Flexor Digitorum Brevis

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal ...