모든 세인트 루이스 심장 분자 공학 연구실에있는 워싱턴 대학의 구성원. 버로우즈는 과학 인터페이스에서 기금 경력 상을 오신 것을 환영합니다 - 1010299을 (JS에).

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저자가 공개하는 게 없다.

절개 난자 바셀린 갭 전압 클램프 기술은 데이터의 신속한 해결이 가능, 낮은 노이즈, 상대적으로 긴 프로토콜 19을 통해 내부 솔루션 및 외부 용액 조성물을 제어하고, 안정된 레코딩을 증가했다. 이러한 장점 떨어져 표준 두 전극 전압 클램프 및 패치 클램프 기술에서이 기술을 설정합니다. 전문 장비가 필요하고 프로토콜이 상대적으로 어려운 있지만 시스템이 최적화되면, 아주 몇 가지 문제가 발생합니다. 이것은 나트륨 (HNA V 1.5) 및 기타 빠르게 활성화 채널의 재현 녹음 할 수 있습니다. 프로토콜에서 가장 중요한 단계는 V1 전극과 난자와 말뚝으로 찌르는 형벌에 최고 실의 배치입니다. 셀과 실 구멍의 가장자리 사이의 밀착되어 레코딩 품질에 큰 영향을 미치고 있습니다. 상부 챔버는 가장 높은 재를 달성하기 위해 셀을 푸시 다운 할 수 있어야합니다난자를 손상시키지 않고 sistance. 이것은 난자 부푼 만들기 요구하고 평평하지만, 셀 품질의 경험과 대가는 파열을 방지하는 최적의 압력을 결정하기 위하여 필요한 것이다. 패스트 기록 일관 세포를 손상시키지 않고 이용 될 수있는 가장 큰 가능한 피펫 개구부의 사용을 필요로한다. 특별한주의는 피펫 팁이 삽입으로 크게 상처를 확장하지 않을 정도로 얕은해야 피펫의 테이퍼에 지불해야한다. 꿰 즉시 막 잠재적 인 독서의 모양에 따라 전극의 발전을 중지, 천천히 그리고 조심스럽게 수행해야합니다. 난자의 세포막에 손상과 완벽한 난자 / 실 물개보다 높은 누설 전류가 발생할 수 있습니다. 높은 누설 전류는 항상 레코딩의 품질을 저하. 따라서, 녹음은 항상 낮은 누설 전류 (바람직하게는 &lt;150 ~ 200 nA의)가 있어야합니다. 앰프의 보상 회로, P / N 온라인 누설 공동 사용rrection, 또는 오프 - 라인 절차는 누설 전류를 보상 할 수있다. 문제 해결은 일반적으로 대부분의 문제를 해결 할 모든 구성 요소가 올바르게 연결 또는 조정을 두 번 검사에 의해 시작해야한다. 준비와 실험 자체 동안 특별한주의는 유출 또는 넘쳐 유체 수준으로 지급하고이 전기적으로 절연되는 것을 의미하는 구획을 연결할 수의 KCl을 결정화해야합니다. 시스템이 예기치 않게 동작하는 경우, 다리와 챔버 사이의 누락 또는 원치 않는 연결을 확인합니다. 에어 한천 교량의 단부에, 한천 교량 내에 기포 또는 셀 아래에 갇혀도 감지하기 어려운 연결 문제를 일으킬 수있다. 수정은, 전술 한 바와 같이, COVG 함께 전압 클램프 형광 측정 (VCF)를 수행하도록 요구된다. 주요 변경은 변경된 목욕 디자인을 사용하는 것입니다. 물 침지 40X 대물 렌즈의 사용을 수용하기 위해현미경, 상부 챔버 욕은 표준 절개 셋업 될 필요보다 커야한다. 다른 측면과 COVG 모드에서 VCF 녹화 장비의 요구는 두 개의 전극 전압 클램프 모드 14 VCF 기록과 유사하다. 앞서 강조한 바와 같이, COVG 기술의 주요 장점은 포유 동물 세포 발현 시스템에서 달성 될 수있는 것보다 훨씬 더 많은 이온 채널을 표현 멤브레인 패치 TEVC에 비해 훨씬 빠르고 정확한 전압 제어이다. 따라서,이 기술은 VCF 높은 시간 해상도와 높은 채널 번호가 모두 검출 신호에 필요한 현재의 연구를 게이팅에 이상적입니다. 원칙적으로 여분의 세포 내 두 솔루션 교환을 할 수 있지만, 환율의 완성도와 속도는 실험의 특정 유형에 대한 몇 가지 제한 사항을 설정합니다. 도 5 및 세포질 하부 참 이온 농도 사이의 평형의 비율에 나타낸 바와 같이BER은 매우 느린 사포닌 permeabilization을 따르고있다. 이온 농도를 변경하는 것은, 따라서 정상 상태에 도달하기 전에 셀 긴 실험에서 고려되어야한다. 환율은 조건에 따라 큰 범위로 변화 할 수있다. 높은 채널 표현, 큰 추진력, 그리고 빠른 속도의 높은 온도의 결과. 우리의 실험에서 세포는 19 ° C로 냉각시키고, 채널 표현은 적당하게, 그리고 IV 프로토콜에 의해 구동되는 순 비용으로 인해 변화하는 현재의 방향을 최소화했다. 이러한 설정에서 일반 COVG 기술은 다양한 패치 - 클램프 구성 열등하다. 세포질 매체의 빠른 교체를 요구하는 COVG 응용 프로그램의 관류의 정맥을 사용할 수있다. 미래에, 내장 된 관류 포트 설계 COVG 챔버 세포 솔루션의 교환 특성을보다 효과적으로 제어 할 수 있습니다.

특수 전압 클램핑 기술은 제어 된 막 전위에서 이온 전류의 기록을 허용한다. 널리 두 전극 전압 클램프 (TEVC)를 사용하고 패치 클램프 기술은 많은 이온 채널의 특성에 대한 신뢰성있는 전기 생리 정보를 제공한다. 그러나, 이러한 방법은 모두 빠른 전압 문 나트륨 채널과 같은 Xenopus의 난자의 세포막에있는 것과 같은 다른 빠른 활성화 채널에 대한 신뢰할 수있는 데이터의 취득을 방지 단점이 있습니다. Bezanilla과 스테파니 연구소는 따라서 난자 2의 절개 바셀린 갭 전압 클램프 기술 (COVG)를 개발했다. 기법은, 나 +, K +, 및 칼슘 2 + 채널 3-8을 기록 널리 적용되어왔다. COVG 기록하는 동안, 이종 단백질 발현 난자의 막을 세 지역으로 나누어 져 있습니다. 이온 전류 데이터로서 난자의 상부 영역으로부터 기록된다정상 영역을 둘러싸 욕을 용이하고 신속하게 변경 될 수있는 명령 전위에 클램프된다. 중간 영역의 상부 영역 (9)과 동일한 전위에 클램프 됨으로써 누설 전류에 대하여 가드. 난자 개구 (절개) 사포닌 용액 또는 정맥의 사용을 통해 발생하는 위치를 하단 영역입니다. 화학적 또는 저부 영역에서 멤브레인의 수동 개방은 접지에 클램프 내부 전위의 제어를 허용하고, 하부 챔버 용액과 인접 셀 내부를 렌더링한다. 상부 챔버의 솔루션 교환이 외부 환경을 변경하는 반면 하부 챔버에 솔루션을 살포는 내부 환경의 특성을 조정할 수 있습니다. <img alt="그림 1" fo:content-width="5in" fo:src="/files/ftp_upload/51040/51040fig1highres.jpg" src="/files/ftp_upload/51040/51040fig1.jpg" width="500px" /> 그림 1. 난자 절개 전압 클램프 욕실 설치 다이어그램. (A) 최고서로 분리 된 세 개의 화장실의 아래로보기. COVG의 챔버의 치수는 그림에 표시됩니다. (B) 테스트 위치에 화장실 설치의 측면보기. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51040/51040fig1highres.jpg" target="_blank">더 큰 이미지를 보려면 여기를 클릭하십시오</a> . COVG 기법의 이점은 낮은 전류 노이즈 (3 kHz에서 1 NA), 외부 미디어의 이온 조성, 내부 용지, 빠른 시간 해상도 (의 붕괴 20-100 마이크로 초 시간 상수를 조절하는 능력의 제어를 포함 용량이 과도), 몇 시간 9 안정 녹음. 단점은 특수 장비를 필요로하며 두 전극 전압 클램프 (TEVC) (10)에 비해 수행하기 더 어려운 것이있다. COVG 접근 방식은 고도의 전문 장비와 복잡한 절차 적 요소가 필요하지만, 그것은 VALU의 인수를 허용 할 수 있습니다수 전기 생리학 데이터. 같은 빠른 반응 속도와 꼬리 전류 4 전류를 게이팅 등이 데이터는, 채널 런 다운 등 다른 전압 클램핑 프로토콜과 관련된 몇 가지 문제없이 녹음 할 수 있습니다. COVG 설정에 약간의 수정은 온도 조절기와 전압 클램프 형광 측정 (VCF)의 사용을 허용 할 수있다. COVG 조립체 내의 전압 클램프 형광 측정 요소의 포함은 동시에 현재 11-13을 촬영하면서 단백질 형태 적 변화를 모니터링하는 기능을 부여하여 데이터 출력을 증강 할 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="515">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">External Solution</td>
			<td style="width:131px;">Brand</td>
			<td style="width:72px;">Catalog Number</td>
			<td style="width:124px;">[Final], weight, or volume</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">N-methyl-D-glucamine (NMDG)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M2004</td>
			<td style="width:124px;">25mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">MES Sodium Salt</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M5057</td>
			<td style="width:124px;">90mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">HEPES</td>
			<td style="width:131px;">Research Products International</td>
			<td style="width:72px;">H75030</td>
			<td style="width:124px;">20mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Calcium hydroxide</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">239232</td>
			<td style="width:124px;">2mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">MES Hydrate</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M8250</td>
			<td style="width:124px;">variable (pH to 7.4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;"></td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Internal Solution</td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">N-methyl-D-glucamine (NMDG)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M2004</td>
			<td style="width:124px;">105mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">MES Sodium Salt</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M5057</td>
			<td style="width:124px;">10mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">HEPES</td>
			<td style="width:131px;">Research Products International</td>
			<td style="width:72px;">H75030</td>
			<td style="width:124px;">20mM</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:189px;">Ethylene glycol-bis(2-aminoethylether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">E4378</td>
			<td style="width:124px;">2mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">MES Hydrate</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M8250</td>
			<td style="width:124px;">variable (pH to 7.4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;"></td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Depolarizing Solution</td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">KCl</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">221473</td>
			<td style="width:124px;">110mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Magnesium chloride</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M8266</td>
			<td style="width:124px;">1.5mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Calcium Chloride</td>
			<td style="width:131px;">Caisson</td>
			<td style="width:72px;">C021</td>
			<td style="width:124px;">0.8mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">HEPES</td>
			<td style="width:131px;">Research Products International</td>
			<td style="width:72px;">H75030</td>
			<td style="width:124px;">10mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;"></td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Pipet Solution</td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">KCl</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">221473</td>
			<td style="width:124px;">3M</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:189px;"></td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:189px;">Saponin Solution</td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Saponin</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">47036</td>
			<td style="width:124px;">0.125g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Internal Solution</td>
			<td style="width:131px;">See above</td>
			<td style="width:72px;"></td>
			<td style="width:124px;">50mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;"></td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;"></td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;"></td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Agar Bridge Solution</td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">N-methyl-D-glucamine (NMDG)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M2004</td>
			<td style="width:124px;">100ml of 1M</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">HEPES</td>
			<td style="width:131px;">Research Products International</td>
			<td style="width:72px;">H75030</td>
			<td style="width:124px;">1.2g</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">MES Hydrate</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M8250</td>
			<td style="width:124px;">variable (pH to 7.4)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">Granulated Agar</td>
			<td style="width:131px;">Research Products International</td>
			<td style="width:72px;">A20250</td>
			<td align="right" style="width:124px;">3%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;"></td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">NMDG Storage Solution</td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">NMDG, HEPES, MES Hydrate solution</td>
			<td style="width:131px;">see above</td>
			<td style="width:72px;"></td>
			<td style="width:124px;">40ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Water</td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;">60ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Name of Material/ Equipment</td>
			<td style="width:148px;">Company</td>
			<td style="width:144px;">Catalog Number</td>
			<td style="width:285px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">High Performance Oocyte Clamp</td>
			<td style="width:148px;">Dagan</td>
			<td style="width:144px;">CA-1B</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Data Acquisition System</td>
			<td style="width:148px;">Axon CNS</td>
			<td style="width:144px;">&#160;Digidata 1440A</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">Oscilloscope</td>
			<td style="width:148px;">Tektronix&#160;</td>
			<td style="width:144px;">TDS 210</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">Rack Power Filter</td>
			<td style="width:148px;">APC&#160;</td>
			<td style="width:144px;">G5</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:149px;">Heating/Cooling Bath Temperature Controller</td>
			<td style="width:148px;">Dagan</td>
			<td style="width:144px;">HCC-100A</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">PC</td>
			<td style="width:148px;">Dell</td>
			<td style="width:144px;">Optiplex 990</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">pCLAMP 10.3 Voltage Clamp Software</td>
			<td style="width:148px;">Molecular Devices, LLC</td>
			<td style="width:144px;">pCLAMP10.3</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:149px;">TMC Vibration Control TableTop Platform</td>
			<td style="width:148px;">TMC</td>
			<td style="width:144px;">64 SERIES</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">TMC Vibration Control Air Table</td>
			<td style="width:148px;">TMC</td>
			<td style="width:144px;"><a name="RANGE!C10">20 Series&#160;</a></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">V1/I Electrode Data Collector</td>
			<td style="width:148px;">Dagan</td>
			<td style="width:144px;">part of CA-1B</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">MX10L Micromanipulator</td>
			<td style="width:148px;">Siskiyou</td>
			<td style="width:144px;">MX10L</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:149px;">Bath/Guard (I/V) Headstage (with appropriate connectors)</td>
			<td style="width:148px;">Dagan</td>
			<td style="width:144px;">part of CA-1B</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">Microscope</td>
			<td style="width:148px;">Omano</td>
			<td style="width:144px;">OM2300S-JW11</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:149px;">Temperature Control Bath</td>
			<td style="width:148px;">Custom or Dagan</td>
			<td style="width:144px;">Custom or HE-204C</td>
			<td style="width:285px;">Custom chamber made from materials from Cool Polymers (D-series). Dagan also provides a prefeabricated stage (HE-204C).</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Custom AgCl Pellet Container</td>
			<td style="width:148px;">Custom</td>
			<td style="width:144px;">Custom</td>
			<td style="width:285px;">Custom machined</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Ag/AgCl electrode, pellet, 2.0 mm</td>
			<td style="width:148px;">Warner</td>
			<td style="width:144px;">E-206</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">External Oocyte Bath</td>
			<td style="width:148px;">Custom or Dagan</td>
			<td style="width:144px;">Custom or CC-1-T-LB</td>
			<td style="width:285px;">Custom machined or purchased from Dagan</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Internal Oocyte Bath</td>
			<td style="width:148px;">Custom or Dagan</td>
			<td style="width:144px;">Custom or CC-TG-ND</td>
			<td style="width:285px;">Custom machined or purchased from Dagan</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:149px;">Capillaries for Agar Bridges and Pulled Electrodes</td>
			<td style="width:148px;">Warner</td>
			<td style="width:144px;">G150T-4</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:149px;">Rotatable Mounts for the Microscope, Micromanipulator, and Bath</td>
			<td style="width:148px;">Siskiyou</td>
			<td style="width:144px;">SD-1280P</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">Fiber-Lite</td>
			<td style="width:148px;">Dolan-Jenner</td>
			<td style="width:144px;">LMI-600</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">Regular Bleach</td>
			<td style="width:148px;">Clorox</td>
			<td style="width:144px;">470174-764</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Xenopus laevis Oocytes</td>
			<td style="width:148px;">Nasco</td>
			<td style="width:144px;">LM535M (sexually mature females)</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:149px;">90 Na+ External Solution</td>
			<td style="width:148px;">See Solutions sheet</td>
			<td style="width:144px;"></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:149px;">10 Na+ Internal Solution</td>
			<td style="width:148px;">See Solutions sheet</td>
			<td style="width:144px;"></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">3 M KCL</td>
			<td style="width:148px;">See Solutions sheet</td>
			<td style="width:144px;"></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">Saponin</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:144px;">47036</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">NMDG Storage Solution</td>
			<td style="width:148px;">See Solutions sheet</td>
			<td style="width:144px;"></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">5mL transfer pipets</td>
			<td style="width:148px;">SciMart</td>
			<td style="width:144px;">GS-52</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:149px;">Modified KCl electrode injector</td>
			<td style="width:148px;">BD</td>
			<td style="width:144px;">309659</td>
			<td style="width:285px;">Plastic syringe tip melted to allow for injection of solution into electrodes. Alternatively, a Microfil by WPI can be purchased.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">Microvaccum</td>
			<td style="width:148px;">Custom</td>
			<td style="width:144px;">Custom</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">Forceps</td>
			<td style="width:148px;">VWR</td>
			<td style="width:144px;">63040-458</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Oocyte Handling Tools (Pipette Pump)</td>
			<td style="width:148px;">VWR</td>
			<td style="width:144px;">53502-222</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Deionized Water Squirt Bottle</td>
			<td style="width:148px;">VWR</td>
			<td style="width:144px;">16649-911</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Vaseline Petroleum Jelly</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:144px;">19-086-291&#160;</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;"></td>
			<td style="width:148px;"></td>
			<td style="width:144px;"></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:149px;">Additional Materials Required for VCF Recordings:</td>
			<td style="width:148px;"></td>
			<td style="width:144px;"></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">VCF Microscope</td>
			<td style="width:148px;">Nikon</td>
			<td style="width:144px;">Eclipse FN1</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Nikon CFI APO 40XW NIR Objective</td>
			<td style="width:148px;">Nikon</td>
			<td style="width:144px;">N40X-NIR</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:149px;">X-Y Translator System for Fixed-Stage Upright Microscopes</td>
			<td style="width:148px;">Sutter Instruments</td>
			<td style="width:144px;">MT500-586</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">External VCF Oocyte Bath</td>
			<td style="width:148px;">Custom</td>
			<td style="width:144px;"></td>
			<td style="width:285px;">Custom machined. The chamber dimensions are 2.7 x 1.9 x 0.4 cm.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Internal VCF Oocyte Bath</td>
			<td style="width:148px;">Custom</td>
			<td style="width:144px;"></td>
			<td style="width:285px;">Custom machined. The chamber dimensions are 1.6 x 1.6 x 0.4 cm.</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:149px;">Modified Temperature Control Bath</td>
			<td style="width:148px;">Custom</td>
			<td style="width:144px;"></td>
			<td style="width:285px;">Custom chamber made from materials from Cool Polymers (D-series). The chamber dimensions of the modified temperature controller bath are 2.7 x 1.9 x 0.3 cm for the horizontal chamber, and 1 x 2.5 x 0.5 cm for the vertical chamber.</td>
		</tr>
	</tbody>
</table>

the xenopus oocyte cut-open vaseline gap voltage-clamp technique with fluorometry

절개 난자 바셀린 갭 (COVG) 전압 클램프 기술은 난자의 이종 이온 채널의 전기 생리학과 운동 특성을 분석 할 수 있습니다. 절개 설정에서 녹음 낮은 크기의 게이트 전류, 빠른 이온 전류의 활성화 및 비활성화를 해결하는 데 특히 유용합니다. 두 전극 전압 클램프 (TEVC) 기법을 통해 주요 장점은 증가 된 클램프 속도, 개선 된 신호대 잡음비, 및 세포 내 및 세포 외 환경을 조절하는 능력을 포함한다. 여기서는 절개 설정 및 프로토콜뿐만 아니라 전압 클램프 형광 측정 기능을 추가하는 데 필요한 변형을 설명하기 위해, (HNA V 1.5), 아프리카 손톱 개구리의 난모 세포에서 발현 인간 심장 나트륨 채널을 이용한다. 이러한 HNA V 1.5 빨리 활성화 이온 채널의 특성은 완전히 whic에서 TEVC를 사용하여 상온 근처에 해결 될 수없는난자의 세포막의 시간 전체가 전압 제어가 어려워 고정된다. 그러나 절개 기술에서 세포막의 단지 작은 부분의 절연은 패치 클램프 기술과 연관된 채널 황폐 방지하면서 정확하게 빠른 반응 속도를 기록 할 필요 급속한 클램핑을 허용한다. COVG 기술, 이온 채널 속도론 및 전기 생리 특성과 함께 추가로 단백질 움직임이 세포 외로 적용 형광체, 유전자에 의해 코딩 형광 단백질의 삽입 또는 부자연 아미노산의 혼입 시스테인 공액 통해 추적되는 전압 클램프 형광 측정을 사용하여 정량 할 수있다 관심 1의 영역으로. 이러한 추가적인 데이터는 형광 분자를 둘러싸는 미세 변화를 통한 단백질의 전압 의존성 구조적 재 배열 대해 운동 정보를 산출한다.

도 4는 디스플레이 사포닌 용액으로서 난자의 투과성의 변화는 난자의 ​​바닥 부에인가된다. 5 사포닌 permeabilization 다음 확산에 의해 세포 내 용액의 교환 율을 보여 도표. 20 ~ 40 분은 정상 상태 2,18에 와서해야합니다. 기록 프로토콜에서 생성 된 그림 6A 쇼 흔적. 그림은 전압 프로토콜 (삽입)에 대한 응답 (P/-8 누출 공제 후) 이온 전류를 보여줍니다. 그림의 각 트레이스는 다른인가 전압을 나타냅니다. 느린 반응 속도와 흔적은 나트륨 채널을 열 수있는 가장 낮은 전위를 나타낸다. 일반적으로, 기존의 방법을 사용하여, 그것을 안쪽 전류 막 탈분극 때문에 이러한 낮은 전위에 대한 전압 제어를 유지하는 것이 곤란하다. 차례로이 탈분극은 긍정적 인 피드백 루프를 만들어 더 많은 채널을 활성화<html> . 절개 기술의 향상 클램프 속도에서도 이러한 어려운 전위 채널을 녹화에 필요한 전압 제어를 가능하게한다. 도 6b는도 6a의 트레이스에서 생성 된 전류 / 전압 곡선을 보여준다. 전압 제어하지 않고 초기 전위에서의 피크 전류 (~ -60 MV)가 과대 평가 될 것이다, 위에서 언급 한. 이 전류 - 전압 관계의 정확한 설명을 방지한다. 도 7은 난 모세포에 대한 전압 클램프 형광 측정 결과의 예를 나타낸다. 형광 신호는 인간의 심장 나트륨 채널 나 V 1.5의 DII 도메인에 위치 (805)에 삽입 시스테인에서 MTS-TAMRA으로 표시 난자에서 기록되었다. _upload/51040/51040fig5.jpg &quot;폭 =&quot;는 500px &quot;/&gt; 그림 5. 내부 솔루션 평형의 세포질 나 + 확산에 의해 농도 변화. 속도의 반응 속도는 세포의 모두 외부 및 내부 측면에 90 밀리미터 나 +를 적용하고 IV 관계마다 2 분을 기록하여 나 + 반전 가능성을 측정하여 평가 하였다. 내부 환경이 완벽하게 대체 된 경우, E 레브 0 MV 것입니다. 사포닌은 난자의 막을 permeabilized 때 시간 측정이 시작되었다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51040/51040fig5highres.jpg" target="_blank">더 큰 이미지를 보려면 여기를 클릭하십시오</a> . <img alt="그림 6" fo:content-width="5in" fo:src="/files/ftp_upload/51040/51040fig6highres.jpg" src="/files/ftp_upload/51040/51040fig6.jpg" width="500px" /> 그림 6. 야생형 나상욱 V 1.5절개 전압 클램핑에서 나트륨 채널의 결과.의 (A) 추적 100 밀리 초에 대한 -120 MV, +40 MV에서의 테스트 펄스 (-120 MV 아래로 -80 값의 유지 가능성과 다른 자극의 전압에서 전류 기록 200 밀리, 그리고 마지막으로 -120 MV에 repolarizing 10 MV 단위). (B) 다음 패널 A의 피크 전류의 전압 의존성, 측정 값이 표시됩니다 +60 최대 펄스의 피크 전류를 나타내는 IV 커브. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51040/51040fig6highres.jpg" target="_blank">더 큰 이미지를 보려면 여기를 클릭하십시오</a> . <img alt="그림 7" fo:content-width="5in" fo:src="/files/ftp_upload/51040/51040fig7highres.jpg" src="/files/ftp_upload/51040/51040fig7.jpg" width="500px" /> 그림 7. 난자에서 기록 나상욱 v 1.5 나트륨 채널 전압 클램프 형광 측정 녹음. (A) 이온 전류인간의 심장 나트륨 채널 나 V 1.5의 DII-S4 도메인의 위치 (805)에 삽입 시스테인에서 MTS-TAMRA으로 표시. -170 MV에서 +70 MV에 이르기까지 전압 펄스는 -120 MV에 전 진동을 다음과 같은 20 밀리의 기간 동안 20 MV 단위로 적용되었다. (B) 관련 형광 신호. 다른 모든 형광 추적은 명확성을 위해 생략된다. ΔF / F는 전압 펄스에 의해 유도 된 형광 강도의 상대적 변화를 나타냅니다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51040/51040fig7highres.jpg" target="_blank">더 큰 이미지를 보려면 여기를 클릭하십시오</a> .

Watch this Scientific Journal Video about The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry at JoVE.com

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

절개 바셀린 갭 접근 방식은 빠른 채널 반응 속도의 높은 해상도와 Xenopus의 난자에서 발현 전압 - 의존성 이온 채널에서 이온 및 게이팅 전류의 낮은 잡음 녹음을 얻는 데 사용됩니다. 사소한 변형에, 전압 클램프 형광 측정은 절개 난자 프로토콜에 연결될 수있다.

<em&gt; Xenopus의</em형광 측정으로&gt; 난자 절개 바셀린 갭 전압 클램프 기술

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

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The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

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18.3K 조회수.  Washington University in St. Louis.  절개 바셀린 갭 접근 방식은 빠른 채널 반응 속도의 높은 해상도와 Xenopus의 난자에서 발현 전압 - 의존성 이온 채널에서 이온 및 게이팅 전류의 낮은 잡음 녹음을 얻는 데 사용됩니다. 사소한 변형에, 전압 클램프 형광 측정은 절개 난자 프로토콜에 연결될 수있다. 

비디오: The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Xenopus Laevis의 Oocytes의 Microinjection

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

연구

생물학

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

의 수정 Xenopus의 oocytes 호스트 전송 방법을 사용하여

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

트렌스 제닉 생산 Xenopus laevis 제한 효소 중재 통합 및 핵 이식에 의해

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

마우스 Oocyte의 Microinjection, 성숙과 Ploidy 평가

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

Single Oocyte Bisulfite Mutagenesis

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription1. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes2. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting.
One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level3. Development of the bisulfite mutagenesis assay has advanced from those originally reported4-6 towards ones that are more sensitive and reproducible7. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment8. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos9. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos10. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos.
Taking clues from agarose embedding of minute DNA amounts including oocytes11, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis12. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos.

Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.

단일 Oocyte의 Bisulfite의 돌연변이 유발

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for ...

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop.
Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle.
The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, &gamma;-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint 4-7. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint 8. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling 9-12.
Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Xenopus egg extract is a useful model system to investigate the DNA damage checkpoint. This protocol is for the preparation of Xenopus egg extracts and DNA damage checkpoint inducing reagents. These techniques are adaptable to a variety of DNA damaging approaches in the study of the DNA damage checkpoint signaling.

사용하여 DNA의 손상 세키의 연구<em&gt; Xenopus</em&gt; 달걀 압축을 풉니 다

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

세포 내 칼슘 측정<sup&gt; 2 +</sup개의 기술하여 인간 정자에서&gt; 변경 : 기존 Fluorometry은 흐름 Fluorometry, 유동 세포 계측법 및 단일 세포 이미징을 중지

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

의 얼굴 이식<em&gt;는 Xenopus laevis의</em&gt; 태아

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including by mechanical forces. Certain TRPV4 mutations cause complex hereditary bone or neuronal pathologies in human. Wild-type or mutant TRPV4 transgenes are commonly expressed in cultured mammalian cells and examined by Fura-2 fluorometry and by electrodes. In terms of the mechanism of mechanosensitivity and the molecular bases of the diseases, the current literature is confusing and controversial. To complement existing methods, we describe two additional methods to examine the molecular properties of TRPV4. (1) Rat TRPV4 and an aequorin transgene are transformed into budding yeast. A hypo-osmtic shock of the transformant population yields a luminometric signal due to the combination of aequorin with Ca2+, released through the TRPV4 channel. Here TRPV4 is isolated from its usual mammalian partner proteins and reveals its own mechanosensitivity. (2) cRNA of TRPV4 is injected into Xenopus oocytes. After a suitable period of incubation, the macroscopic TRPV4 current is examined with a two-electrode voltage clamp. The current rise upon removal of inert osmoticum from the oocyte bath is indicative of mechanosensitivity. The microAmpere (10-6 to 10-4 A) currents from oocytes are much larger than the subnano- to nanoAmpere (10-10 to 10-9 A) currents from cultured cells, yielding clearer quantifications and more confident assessments. Microscopic currents reflecting the activities of individual channel proteins can also be directly registered under a patch clamp, in on-cell or excised mode. The same oocyte provides multiple patch samples, allowing better data replication. Suctions applied to the patches can activate TRPV4 to directly assess mechanosensitivity. These methods should also be useful in the study of other types of TRP channels.

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

To complement commonly used methods to study TRPV4&#8217;s, two methods are described: Its mechanosensitivity can be studied by Ca2+-aequorin luminometry in transgenic yeast upon hypo-osmotic challenge. It can also be examined in TRPV4-RNA injected Xenopus oocytes by whole-cell two-electrode voltage clamp or patch clamp in on-cell or excised mode.

효모 Luminometric 및<em&gt; Xenopus의</emTRPV4의 분자 Mechanosensitivity의&gt; 난자 전기 생리 시험

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Xenopus의 번역 손상을 식별 할 수있는 모델로 laevis의

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...