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>

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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+A2:D32D42A2:D31AA2:D29</td>
			<td>Life Technologies</td>
			<td>11995-073</td>
			<td>Dissection Media 
			Dilution: 50ml +100g BSA</td>
		</tr>
		<tr>
			<td>Collagenase, Type II</td>
			<td>Life Technologies</td>
			<td>17101-015</td>
			<td>Fiber Digestion 
			Dilution: 160mg / 4ml DMEM (stock 40mg/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 
			Dilution: 146 mM NaCl 
			5 mM&#160;&#160; KCl 
			2 mM&#160;&#160; CaCl2 
			1 mM&#160;&#160; MCl2 
			10mM HEPES 
			pH7.4 in H2O</td>
		</tr>
		<tr>
			<td>1ml Pipettes</td>
			<td>Axygen</td>
			<td>T-1000-C-R</td>
			<td>Digestion</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Razor Blades</td>
			<td>Personna</td>
			<td>94-120-71</td>
			<td>Digestion</td>
		</tr>
		<tr>
			<td>Precision Glide 30G needle</td>
			<td>BD Biosciences</td>
			<td>305128</td>
			<td>Dissection</td>
		</tr>
		<tr>
		</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 
			Dilution: 100mg / 50ml DMEM</td>
		</tr>
		<tr>
			<td>Falcon 60mm dishes</td>
			<td>Fisher Scientific</td>
			<td>08772B</td>
			<td>Dissection- used to hold sylgard</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Clay</td>
			<td></td>
			<td></td>
			<td>Electroporation</td>
		</tr>
		<tr>
			<td>Stimulator&#160;</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>
		</tr>
		<tr>
			<td>Precision Glide 27G needle</td>
			<td>BD Biosciences</td>
			<td>305136</td>
			<td>Electroporation</td>
		</tr>
		<tr>
		</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 
			Dilution: Add 160ul PBS (1X) to 40&#181;l 5x stock</td>
		</tr>
		<tr>
			<td>1cc U-100 Insulin Syringe (28G1/2)&#160;</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>35mm glass bottom Microwell dish</td>
			<td>MaTek</td>
			<td>P35G-1.5-14-C</td>
			<td>Microscopy</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>FM 4-64</td>
			<td>Life Technologies</td>
			<td>T-13320</td>
			<td>Microscopy 
			Dilution: Final 2.5 ug/ml</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>FM 1-43</td>
			<td>Life Technologies</td>
			<td>T-35356</td>
			<td>Microscopy 
			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>
		<tr>
		</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

Isolation of Genomic DNA from Mouse Tails

마우스 뒷면에서 게놈 DNA의 분리

연구

생물학

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

Mycobacteria의 Electroporation

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

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 ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

DNA의 손상 응답 단백질의 2와 3 차원 라이브 셀 이미징

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

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 ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

단백질-DNA 상호 작용의 연구에 대한 단일 분자 조작 및 이미지를 결합

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

비례 생체 분자 비콘을 사용하여 살아있는 세포의 단일 엔지니어링 RNA 성적 증명서의 실시간 영상

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

프로토 타입 소형 흐름 사이토와 동반자 미세 유체 혼합 기술의 감소 - 중력 환경 하드웨어 데모

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

레트로 바이러스 통합 사이트의 증폭, 차세대 시퀀싱 및 게놈 DNA 매핑

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...