The authors would like to acknowledge Wensheng Liu for invaluable help with cell imaging. This work was supported by grants from the&#160;US National Institutes of Health (R01 CA182467), American Cancer Society (RSG-09-252-01-RMC), Lynn Sage Foundation, and A Sister&#8217;s Hope Foundation.

EMT 유도의 1 세포 배양 참고 : EMT는 TGFβ의 치료 또는 상피 세포에서 전사 인자 트위스트, 달팽이, 또는 Zeb1 / 2의 이소성 발현에 의해 유도 될 수있다. 불후의 인간의 유방 상피 세포 (HMLE / 트위스트 - ER 박사는 J 양의 선물, UCSD) 9,11,29의 트위스트 - ER 융합 단백질의 발현을 통해 유도 EMT 시스템은이 프로토콜에 설명한다. 4 hydroxytamoxifen (TAM) 처리시, 융합 단백질 트위스트-ER은 12-14일 9,11,29에 전체 EMT 전환에 전사 및 성과를 위해 핵 옭. 같은 HMLE / 달팽이-ER, HMLE / TGFβ 및 MCF10A / TGFβ 등의 추가 EMT 유도 시스템은 이전 출판물 11,30에서 찾을 수 있습니다. <ol><li> 5 % CO 2와 37 ° C 배양기에서 무 혈청 유방 상피 세포 성장 매체 (MEGM)에서 HMLE / 트위스트 - ER 세포를 유지한다. 통로 세포는 2-3 일마다 그들은 80 %의 합류에 도달하면. 5 % 혈청 / DMEM과 트립신을 비활성화 한 후 5 ~ 10 분 동안 37 ° C에서 0.15 % 트립신으로 세포를 품어합니다. 세포를 스핀 다운 및 MEGM에서 그들을 재현 탁. 1 내지 4의 비율로 새로운 조직 배양 접시에 접시 세포. </li><li> 200 μM 솔루션을 만들기 위해 에탄올에 TAM을 녹인다. -20 ° C에서 빛과 상점에서 TAM를 보호합니다. 20 nM의 TAM의 최종 농도를 희석하기 위해 10,000 : 사용 전에 갓 1에서 MEGM 매체를 추가로 TAM. </li><li> EMT의 유도를 들어, 중복에 10cm 조직 배양 접시에 1.6 × 10 6 HMLE / 트위스트 - ER 세포를 접시. 20 nM의 TAM 함유 MEGM 미디어와 문화 세포. </li><li> 중복에 10cm 접시에 플레이트 1.6 × 10 6 HMLE / 트위스트 - ER 세포와 비 TAM 처리 제어와 같은 0.01 % 에탄올로 치료 세포. </li><li> 두 일 배양 한 후, 세포의 형태 학적 변화를 기록하기 위해 10 배 배율의 광학 현미경 사진을 찍을. </li><li> 제어 또는 TAM 처리의 한 접시에서 매체를 기음세포. RNA 분리 및 단백질 분석 RIPA 버퍼 내의 다른 절반 RNA 용해 완충액 플레이트의 절반을 폐기하여 차가운 PBS와 세포 및 세포 수확을 씻는다. </li><li> 중복에 10cm 접시에 재 도금 1.6 × 10 6 세포에 의한 제어 또는 TAM 처리 된 요리에서 통과 세포. 연속 20 nM의 TAM 또는 차량 세포를 취급합니다. </li><li> 반복 차량 처리 된 대조군 세포는 조약돌 모양의 상피 세포 형태를 유지하는 반면, 어떤 시간에, TAM 처리 트위스트 - ER 세포, 방추형 중간 엽 형태를 보여, 14 일까지 5 회 매일 7 단계를 반복합니다. </li></ol> EMT의 2 특성 참고 : EMT의 완료가로 표시됩니다 : 스핀들 모양의 섬유 모세포와 같은 외관 조약돌 모양의 상피 형태에서 (1) 셀 형태 학적 변화; (2) 세포 - 세포 접합에서 E-cadherin의 현지화 패, 손실에 의해 정의 EMT 마커 및 (3) 식 스위치,상피 마커 및 중간 엽 마커의 이득. 세포 형태 학적 변화는이 절에 설명되어 세포 접합부에서 E-cadherin의 제 1 면역 형광 검출에 설명, EMT 유도의 시간 경과시 10 배 배율의 광학 현미경에 의해 캡처됩니다. EMT 마커의 발현은 발현 수준에 의해 검출된다. 일반적인 상피 마커는 E-cadherin의, γ-catenin이, 그리고 occludin, 그리고 중간 엽 마커 피브로넥틴, N-cadherin의 및 멘틴을 포함한다. 면역 블로의 일반 절차는 4 절에 설명되어 있습니다. <ol><li> TAM 처리, 시드 24 - 웰 플레이트에 넣고 잘 12mm 원형 유리 커버 슬립에 1 × 105 세포에서 12 일. </li><li> 미리 예열 PBS와 시드, 흡인 매체 및 세척 세포 후 48 시간. </li><li> 15 분 동안 미리 예열 4 % 파라 포름 알데히드로 세포를 고정합니다. </li><li> PBS로 세포를 세 번 씻으십시오. </li><li> 15 PBS에서 0.2 % 트리톤 X-100으로 세포를 부화RT에서 분. </li><li> PBS로 세포를 세 번 씻으십시오. </li><li> 비특이적 결합 차단 RT에서 1 시간 동안 5 % BSA / PBS에서 세포를 배양한다. </li><li> 4 ° C에서 1:50 희석 1 %의 BSA / 가습 챔버 O에서 PBS / N에 차 항체 E-cadherin의와 세포를 품어. 참고 : 차 항체의 희석 범위는 1:50 일 사이에 일반적입니다 : 200, 각 항체에 대한 최적의 희석은 실험적으로 결정되어야한다. </li><li> 차 항체를 가만히 따르다하고 PBS로 세포를 세 번 씻는다. </li><li> 실온에서 1 시간 동안 500 희석 : 1에 형광 표지 된 이차 항체와 세포를 품어. 에서이 단계에서 빛으로부터 세포를 보호합니다. </li><li> 차 항체를 가만히 따르다하고 PBS로 3 회 씻는다. </li><li> 를 10 분 동안 0.1 g / ㎖ DAPI 또는 훽스트와 얼룩 셀. </li><li> PBS로 3 회 세포를 씻어. </li><li> 마운트 매니큐어와 장착 매체의 드롭 및 밀봉 된 커버로 된 커버. </li><li> 세포를 관찰하고 수행63X 형광 현미경 이미지. </li></ol> 추가 항체로 염색하는 EMT 표현형을 확인하기 위해 수행 할 수 있습니다. 예를 들어, N-cadherin의 및 α-평활근 액틴은 중간 엽 표현형 29,31,32 스테인드 할 수 있습니다. 골격 구조의 개편은 F-액틴 11 phalloidin으로 세포를 염색하여 모니터링 할 수 있습니다. 또한, 세포 운동성 및 세포 사멸에 대한 저항성 분석법 EMT (11)의 특성을 조사하기 위해 수행 될 수있다. QRT-PCR 분석 실험을 사용하여 검출 스플 라이스 동종의 3 <ol><li> 프라이머 디자인 NOTE : 프라이머 쌍은 신중 구별 동종 접합을 증폭하도록 설계되어야한다. 스킵 엑손, 가장 일반적으로 관찰 대안 접합 이벤트, 프라이머 디자인 전략을 설명하기 위해 예로서 여기에 도시되어있다.도 1a는 가변 엑손로서 제 엑손으로, 네 엑손 사전의 mRNA를 도시한다. 엑손의 포함 3 결과엑손 3의 스킵 반면 긴 스플 라이스 이소 형의 생산은, 스플 라이스 짧은 이소 형을 생성한다. 프라이머는 엑손-3-이소 포함하고 엑손-3-이소 스킵를 구별하고,이 전체의 mRNA 전 사체를 검출하도록 설계 될 필요가있다. <ol><li> 엑손 개재물의 검출을 위해, 신중하게 가변 엑손 포함 이소 형의 비 증폭을 허용 변수 엑손 3 및 인근 항시 엑손 4 내부 역방향 프라이머 안에 순방향 프라이머를 설계한다. 순방향 설계 및 게놈 DNA 또는 전-mRNA의 PCR 증폭에서 제품을 피하기 위하여 동일한 변수 엑손 내의 역방향 프라이머하지 않도록주의. </li><li> 특히 엑손 건너 뛰는 이벤트, 디자인을 증폭하려면 변수 엑손 3가 포함 된 경우, 그렇지 않으면 파괴 될 것이다 구성 적 엑손 2 엑손 4의 교차점에 걸쳐 프라이머 중 하나. 이와 같이 항시 적 엑손 4 안에 다른 프라이머를 설계, PCR 제품 변수 엑손 3 exclud 때만 생성된다ED 및 엑손 2, 엑손 사이어서 서로 결합 항시. </li><li> , 유전자의 전체 전 사체를 검출이 항시 엑손 1 및 2에서 프라이머를 설계하기 때문에, PCR 생성물은 모든 아형에서 증폭된다. </li><li> 반드시 모든 프라이머에 대한 융해 온도 (TM)는 약 55 ° C, 각 프라이머의 길이는 약 18-22 뉴클레오타이드이다 확인하고, PCR 생성물의 크기는 80 내지 150 염기쌍이다. </li></ol></li><li> RNA 추출 (재료의 표 참조) 총 RNA 분리 키트를 사용하여 RNA를 추출합니다. <ol><li> 350 ㎕의 RNA의 용해 완충액에 세포를 수집한다. </li><li> 해물을 70 % 에탄올 350 μl를 첨가하고 잘 섞는다. </li><li> RNA 정제 컬럼에 샘플을 전송합니다. </li><li> 1 분 및 폐기의 흐름을 통해 10,000 XG에 원심 분리기. </li><li> RNA의 세척 버퍼 II 회 RNA 세척 버퍼 I의 500 μL와 배의 열을 씻으십시오. </li><li> 잔류 RNA 세척 버퍼를 제거2 분 동안 최대 속도에서 원심 분리에 의해 컬럼으로부터 II. </li><li> , 새로운 1.5 ㎖의 튜브에 열을 이동시켜 열 행렬의 중심에 50 μL 클레아없는 물을 첨가하고 최대 속도로 원심 분리하여 RNA를 용출시킨다. </li></ol></li><li> 첫 번째 가닥의 cDNA 합성 <ol><li> UV 흡수 측정에 의한 농도와 RNA의 품질을 결정합니다. 참고 : 좋은 품질의 RNA는 약 2.0의 260 나노 미터 / 280 nm의 흡광도 비율을 가지고 있어야합니다. </li><li> 20 μL의 최종 볼륨에서 250 NG 총 RNA, 0.05 μg 랜덤 헥사 머 프라이머, 50 pmol의 MgCl2를, 10 pmol의의의 dNTP, 2 ㎕의 5 × GoScript 버퍼, 1 μL RT 효소를 포함하는 역전사 효소 (RT) 반응을 설정합니다. </li><li> RT 효소를 불 활성화하기 위해 5 분 동안 70 ° C에서 배양 한 다음 1 시간, 42 ° C에서 RT 반응을 수행합니다. </li></ol></li><li> 실시간 PCR <ol><li> 10 μL의 SYBR 녹색 마스터 믹스로 구성되어 20 ㎕의 반응을 설정,0.1 ~ 1.0 ㎕의 cDNA를 템플릿, 5 pmol의 앞으로 및 역방향 프라이머. </li><li> 다음 단계의 40주기 실시간 PCR을 실행하여 95 ° C 변성을 10 초, 10 초 동안 58 ° C 어닐링, 30 초 동안 60 ° C의 확장을 위해. 세중에서 PCR 반응을 수행합니다. </li><li> 해리 곡선을 확인하고 하나의 피크가 각각의 프라이머 세트에 대한 관찰되어 있는지 확인합니다. </li><li> 증폭 곡선의 이차 미분 값을 계산하여 정량화 사이클 (Cq와) 값을 얻습니다. </li><li> &#39;델타 델타 Cq와 (ΔΔCq)&#39;다음의 화학식으로 나타낸 바와 같이 방법을 사용하여 유도되지 않은 샘플과 비교하여 샘플에서 유도 된 대상의 mRNA의 상대적 양 (RQ)를 계산한다. 참고로, 같은 GAPDH 또는 TBP 등의 하우스 키핑 유전자를 사용합니다. </li></ol></li></ol><img alt="식 (1)" fo:content-width="3in" src="/files/ftp_upload/51845/51845eq1.jpg" /> 더 정확하게 스플 라이스 동종의 상대 값을 계산하려면 t그는 고려되어야 여러 참조 유전자의 사용합니다. 결과 Pfaffl 외 33,34 설명 변성 절대치 정량 방법을 사용하여 분석 할 수있다. 단백질 수준에서 스플 라이스 동종의 4 시험 <ol><li> 100 ㎕의 얼음처럼 차가운 RIPA 버퍼에 세포를 수집합니다. </li><li> 약 10 분 동안 얼음에 해물을 설정합니다. </li><li> 4 ° C에서 10 분 동안 14,000 XG에 원심 분리하고, 상층 액을 수집합니다. </li><li> 브래드 포드 분석에 의해 단백질 농도를 측정한다. </li><li> SDS 로딩 버퍼에 단백질 시료를 희석하고 10 % SDS-PAGE 젤에 단백질의 20 ~ 40 μg을로드합니다. </li><li> 1.5 시간 동안 20mA에서 SDS-PAGE 젤을 실행합니다. </li><li> 85 V.에서 3 시간 동안 4 ° C에서 젖은 전송 시스템에서 PVDF 멤브레인에 전송 단백질은 목적 단백질의 분자량에 따라 전송 시간을 조정합니다. </li><li> 30 분 실온에서 1 시간에 5 % 무 지방 우유에 차단 막. </li><li> 막 위스콘신 품어4 ° CO / N에 차 항체를 일. (200)와 1 : 5000, 실험적 각 항체에 대한 최적의 희석을 결정 차 항체 희석의 범위는 일반적으로 (1) 사이에있다. 이 프로토콜에서 사용되는 항체의 희석은 &quot;특정 시약 및 장비의 테이블&quot;에서 찾을 수있다. <ol><li> 스플 라이스 아형을 검출하기 위해, 특히 특정한 이소 형을 인식하는 항체를 사용한다. 대안 적으로, 구성 적 코딩 엑손 영역의 에피토프를 인식하는 항체를 사용하여 서로 다른 크기의 단백질을 기반으로 동시에 접합 아형을 검출한다. </li><li> 동시에, EMT 마커에 대한 발현 수준에 의해 EMT를 모니터링 할 수 있습니다. 중간 엽 마커로 사용하여 E-cadherin의, γ-catenin이, 상피 마커로 occludin 및 피브로넥틴, N-cadherin의 및 멘틴. </li></ol></li><li> 5 분 간격으로 TBS-T (50 mM 트리스, 150 mM의 염화나트륨, 0.05 % 트윈 20, 산도 7.4)로 세척 막 세 번. </li><li> HRP-결합 된 보조 개미와 멤브레인을 품어실온에서 1 시간 동안 만 희석 : 1 ibody. </li><li> 5 분 간격으로 TBS-T로 세척 막 세 번. </li><li> 화학 발광 검출 계와 단백질을 시각화하고 오토 라디오 그래피 막에 막을 노출. </li></ol>

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The authors declare that they have no competing financial interests.

여기에 설명 된 절차를 유도 EMT 모델에서 스 플라이 싱의 검출을 가능하게한다. 스플 라이스 이소 형의 발현의 이러한 동적 변화가 EMT의 시간 경과에 걸쳐 캡처 할 수있다. UN 관련 세포주 구별 유전 배경 부당 대체 스 플라이 싱에 영향을 미칠 수 있기 때문에이 방법은 다른 스 플라이 싱의 비교를위한 상이한 epithelial- 또는 간엽-나타내는 세포주의 사용에 이점을 갖는다. 그러나, EMT의 성공적인 유...

상피 - 간엽 전환 (EMT)의 배 발생시 기관의 형태 형성 및 조직 리모델링을 구동하는 발달 프로그램입니다. 비정상적으로 활성화되면, EMT는 종양 전이 및 장기 섬유화 1,2를 촉진한다. 강제적 인 연구 cobble-의 유실있는 adherens 접합 단백질 E-카드 헤린의 발현을 억제 그러한 트위스트, 달팽이 및 ZEB 여러 전사 인자,,,로 정의, EMT 과정에서 전사 조절의 중요성을 설명 하였다 상피 형태와 스핀들 모양의 중간 엽 표현형 3-8의 이득과 같은 돌. RNA를 게놈 차원의 분석을 통해 최근의 연구 접합 패턴 상피 또는 중간 엽 표현형 9, 10 중 하나와 관련된 유전자의 그룹이 존재하는 것으로 나타났습니다. 우리가 실험실에서 작업 기능적 선택적 스 플라이 싱 (alternative splicing)와 EMT 연결. 세포 표면 부착 분자 CD44을 연구함으로써 CD44의 altern 시연극상의 접합이 긴밀하게 EMT 동안 규제하고, 더 중요한 것은, 인과 적으로 전환하는 CD44 스플 라이스 이소 11 EMT에 기여한다. 대안 12-14을 접합하는 인간의 다중 엑손 유전자의 95 %까지로 대체 접합은 유전자 조절의 광범위하고 보존 모델을 나타냅니다. 단일 유전자로부터 다중 단백질 제품을 생성함으로써, 대안적인 스 플라이 싱은 인간 게놈에 또 다른 층의 복잡성을 추가 단백질 다양성 필수기구를 구성한다. 따라서, 선택적 스 플라이 싱 (alternative splicing)의 조절 이상이 잠재적으로 인간의 질병을 일으키는 원인이 심오한 생물학적 효과가 발생할 수 있습니다. 사실, 질병에서 벗어난 대안으로 연결할 수는 spliceosome 기계를 코딩하는 유전자의 돌연변이는 일반적으로 골수 형성 이상 증후군 26 ~ 28에서 발견되는 최근의 연구 결과를 포함, 10 년 이상 15 ~ 25에 설명되어 있습니다. 따라서, 다르게 스 플라이 싱 된 난의 검출을위한 신뢰성있는 방법을 개발soforms는 EMT 등 다양한 생물학적 과정의 연구에 매우 중요하다. 여기서 우리는 유도 EMT 모델을 사용하여 대체 스 플라이 싱의 변화를 검출하는 프로토콜을 제공한다. 스플 라이스 이소 폼을 PCR 프라이머를 설계 및 검출하기위한 방법이 EMT 대체 스 플라이 싱 동안의 연구뿐만 아니라 다른 생물학적 과정에서 대체 스 플라이 싱의 연구뿐만 아니라 적합하다. EMT 동안 대체 스 플라이 싱을 더 조사하면, 따라서 암 전이를 치료하기위한 효과적인 전략의 개발을 촉진 EMT 및 종양 전이의 메커니즘을 이해하기 위해 필수적이다.

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		<tr height="21">
			<td height="21" style="height:21px;">4-hydroxytamoxifen</td>
			<td>Sigma</td>
			<td>H7904</td>
			<td>Make stock solution by dissolving in ethanol to 200&#956;M, and keep at -20&#8451; protected from light.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E.Z.N.A. Total RNA Isolation kit</td>
			<td>Omega Bio-Tek</td>
			<td>R6731</td>
			<td>Total RNA isolation kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GoScript Reverse Transcription System</td>
			<td>Promega</td>
			<td>A5001</td>
			<td>Reagent for qRT-PCR assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GoTaq qPCR Master Mix</td>
			<td>Promega</td>
			<td>A6002</td>
			<td>Reagent for qRT-PCR assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LightCycler 480 Real-Time PCR System</td>
			<td>Roche</td>
			<td></td>
			<td>Equipment for qRT-PCR assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD44 antibody</td>
			<td>R&#38;D Systems</td>
			<td>BBA10</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E-cadherin antibody</td>
			<td>Cell Signaling Technology</td>
			<td>4065</td>
			<td>1:2500 dilution for immunoblotting; 1:50 dilution for immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#947;-catenin antibody</td>
			<td>Cell Signaling Technology</td>
			<td>2309</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">occludin antibody</td>
			<td>Santa Cruz Biotechnology Inc.</td>
			<td>sc-5562</td>
			<td>1:500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fibronectin antibody</td>
			<td>BD Transduction Laboratories</td>
			<td>610077</td>
			<td>1:5000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-cadherin antibody</td>
			<td>BD Transduction Laboratories</td>
			<td>610920</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vimentin antibody</td>
			<td>NeoMarkers</td>
			<td>MS-129-p1</td>
			<td>1:500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GAPDH antibody</td>
			<td>Millipore Corporation</td>
			<td>MAB374</td>
			<td>1:10000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amasham ECL Western blotting detection reagent</td>
			<td>GE Health Life Science</td>
			<td>RPN2209</td>
			<td>Chemiluminescence system</td>
		</tr>
	</tbody>
</table>

detection of alternative splicing during epithelial-mesenchymal transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and &#947;-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.

상술 한 절차는 EMT 동안 대체 스 플라이 싱을 검출하는 강력한 방법을 제공한다. 트위스트에 의한 EMT 동안 CD44 스플 라이스 이소 전환의 대표적인 결과는 예를 들어 다음과 같습니다. HMLE / 트위스트 - ER 세포에서 트위스트에 의한 EMT는 가늘고 긴 섬유 모세포 표현형 상피 표현형 같은 자갈 돌에서 전환 (그림 2A), 상피 마커 E-cadherin의, γ-catenin이와 occludin과의 부재?...

Watch this Scientific Journal Video about Detection of Alternative Splicing During Epithelial-Mesenchymal Transition at JoVE.com

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

상피 - 간엽 전환시 선택적 스 플라이 싱 (alternative splicing)의 검출

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various ...

detection-alternative-splicing-during-epithelial-mesenchymal

Research

JoVE Journal

Biology

12.8K 조회수.  Northwestern University Feinberg School of Medicine. Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

비디오: Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

마우스에 대한 진한 / 라이트 전환 테스트

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

연구

생물학

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

단백질 Ubiquitination의 감지

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

Mycoplasma의 최적화된 PCR 기반의 감지

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

미토콘드리아 절연 마우스 중심부에 투과율 전환 기공 영업의 다중 매개 변수 측정

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

헤어 재생시 상피 - 중간 엽 시그널링의 빠른 유전 분석

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

간엽 전환을 유도하고 상피의 분석

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

인간 다 능성에 대한 대안 문화는 전지 생산, 유지 보수 및 유전 분석을 줄기

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually untranslated &#8212; first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.

방법은 쥐 Bcrp1의 대체 발기인 사용 및 전사 규정을 발견 할 수

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

TEM 분석을위한 낮은 셀 번호와 샘플 준비를위한 다른 방법

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-&#946;, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-&#946; and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&amp;#946; Signaling

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-&#946; in MS-1 endothelial cells.

내 피 엽 전환 성장 인자-β 신호 변환에 의해 유도 된의 분자 분석

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...