RNA의 지방화에 대한 우리의 작업은 KLM하는 NIH (R01GM071049)에서 부여로 지원됩니다.

1 부 : 찬란 라벨 mRNA의 전사. <ol><li> RNA의 현지화 요소 또는 기타 관련 시퀀스를 포함하는 플라스미드 DNA를 Linearize 및 DEPC - H 2 O. 처리 1 μg / μl에 resuspend DNA 템플릿은 T7, SP6 또는 T3 RNA 효소에 의해 해독을위한 업스트림 발기인 사이트가 있어야합니다. </li><li> 멸균 1.5mL 튜브에 다음 시약을 추가합니다 : <table><tbody><tr><td> A. 10X TX 버퍼 (M &amp; M 참조) </td><td> 2 μl </td></tr><tr><td> B. 20X 모자 / NTP 믹스 (M &amp; M 참조) </td><td> 1 μl </td></tr><tr><td> C. 1 ㎜ 알렉사 플루어 546-14 - UTP (Invitrogen) </td><td> 1 μl </td></tr><tr><td> D. UTP, [α - 32 P] (1 μCi / μl) (퍼어킨 엘머) </td><td> 1 μl </td></tr><tr><td> E. DEPC - H 2 O </td><td> 11 μl </td></tr><tr><td> F. 0.2 M DTT </td><td> 1 μl </td></tr><tr><td> G. RNasin (Promega) </td><td> 1 μl </td></tr><tr><td> H. 선형 템플릿 DNA </td><td> 1 μl </td></tr><tr><td> I. RNA 효소 (Promega) </td><td> 1 μl </td></tr></tbody></table></li><li> 부드럽게 시약을 혼합하고 (10 초. microcentrifuge에서) 잠깐 원심 분리기. </li><li> 형광단의 photobleaching 방지하기 위해 알루미늄 호일로 튜브를 덮고 37 ° C에서 2~4시간을위한 부화. </li><li> 템플릿 DNA이 저하될 1 μl 1 MG / ML RNase 무료 DNase (Promega)를 추가합니다. </li><li> 37 15 분 품어 ° C. </li><li> 반응을 중지 79 μl 20 MM EDTA (에틸렌 다이아 민 테트라 초산) (산도 8.0)를 추가합니다. </li><li> &quot;입력&quot;과 저장라는 별도의 튜브, 1 μl를 제거합니다. </li><li> 전체 길이 mRNA를 제외한 동안 비법인 세포핵을 포함하는 1 ML G50 칼럼을 통해 스핀 반응 (재료 및 방법 참조). </li><li> 에탄올 침전으로 RNA를 집중 : <ol class="lalpha"><li> 250 μl 100 % 에탄올, 10 μl은 7m CH 3 COONH 4, 1 μl 캐리어 RNA (tRNA 효모, 10 μg / μl) 또는 1 μl 글리코겐 (20 MG / ML) 추가합니다. </li><li> 잘 믹스하여 -80에서 고체까지 동결 ° C&gt; 30 분 또는 15 분 동안 드라이 아이스에. </li><li> 15 분 가만히 따르다 뜨는 위해 microcentrifuge의 최대 속도로 회전. </li><li> 75 % 에탄올 150 μl로 씻으십시오. </li><li> microcentrifuge, 가만히 따르다 뜨는의 최대 속도로 3 분 스핀. </li><li> 모든 에탄올이 증발했다는 보장, 3 분 동안 37 ° C 펠릿 건조. </li></ol></li><li> 11 μl DEPC - H 2 O의 Resuspend 및 &quot;포함&quot;으로 표시 별도의 튜브, 1 μl를 제거합니다. </li><li> %의 설립을 결정하고 (재료 및 계산 방법을 참조하십시오) 50 nm의로 RNA를 희석. </li><li> RNA는 동결 / 해동 사이클을 피하기 위해 단일 ​​사용 5 μl aliquots에 냉동이어야하며 -80 ° C.에 몇 주 동안 저장할 수 있습니다 </li></ol> 2 부 : Microinjection 준비. <ol><li> RNA 준비 : RNA (50 NM)의 나누어지는를 해동하고, 70 RNA를 변성 ° C를 3~5분하십시오. 모든 입자를 제거하고, 얼음을 유지하는 최대 속도에서 실내 온도 microcentrifuge에서 10 분 스핀. </li><li> Oocyte 준비 : <ol class="lalpha"><li> 수술 Xenopus laevis 여성 (Nasco)에서 난소를 제거합니다. </li><li> Collagenase 솔루션 25 ML을 포함한 50 ML 원뿔 관에 Xenopus laevis의 난소의 트림 조각 (재료 및 방법 참조). </li><li> 15 분 동안 18 ° C에서 부드럽게 흔들거나 oocytes까지 가시 난소에서 구분됩니다. 배치 변화로 배치로 인해, collagenase 처리 시간은 다를 수 있으며 defoliculation을 보장하기 위해 육안 검사를 통해 신중하게 모니터링해야합니다. </li><li> oocytes가 튜브에 정착하도록 허용, 솔루션을 제거하고 (재료 및 방법 참조) MBSH 버퍼로 oocytes 씻으십시오. </li><li> 세 세척 총 두 번 더 씻어 반복합니다. 이 oocyte 격리 프로토콜 코헨 외., 2009에 대한 자세한 비슷합니다. </li><li> 표준 해부 현미경 MBSH 버퍼에 수동 정렬 스테이지 III / IV oocytes (뒤몽, 1972). 단계 IV의 oocytes는 안료와 약간 크고 스페클드하는 동안 스테이지 III의 oocytes 완전히 불투명하지만 흰색입니다. 약간 투명 Oocytes 완전히 색소 또는 전시 편광 안료 배포가 너무 오래, 너무 젊은 oocytes 있습니다. </li></ol></li><li> 니들 준비 : ~ 0.05 mm의 외경에 바늘을 당겨와 베벨. (우리는 드루먼드 과학 3.5 인치 모세관을 사용하여 (주문 # 3 - 000-203 - G / X), 셔터 악기 (주) micropipette 풀러와 WPI 주식 beveller.) </li></ol> 파트 3 : Microinjection <ol><li> 사출 당 2 NL를 제공하기 위해 DEPC - H 2 O와 바늘을 보정합니다. 우리는 앞에서 가스 구동 microinjector을 (재료 및 방법 참조)를 사용하여 우리의 바늘을로드하고 마이크로 미터를 사용하여 드롭 크기를 보정. </li><li> 바늘로 RNA를로드합니다. </li><li> 장소 MBSH 버퍼를 포함하는 주입 그릇에 oocytes를 정렬. 우리는 검은 거품 고무의 레이어와 함께 접시에 microinject. 창백한 oocytes는 흰색 backgrou에 잘 눈에 띄는ND. </li><li> 조심스럽게 50 NM에서 RNA 2 NL 각 oocyte를 삽입. </li><li> , RNA DEPC - H 2 O와 바늘을 씻어하고, 주사에 대한 다음 RNA를 부하를 추방. </li></ol> 4 부 : Oocyte 문화 <ol><li> 멸균 24 잘 플레이트 (시그마 알드리치)의 우물에 플레이스 oocytes. 물론 당 천 oocytes에 우리 문화를 백업하십시오. </li><li> 버퍼를 제거하고 400 μl O ocyte C ulture M edium로 교체 (OCM, 재료 및 방법 참조) 잘마다. 문화 중에 촉촉한 환경을 유지하기 위해 서부 유럽 표준시 종이 타월로 빵빵한 플라스틱 컨테이너 안의 문화 플레이트 플레이스. </li><li> 18 ° C 8 및 48 시간 사이에 이르기까지 시간이 지점에 대한 oocytes를 품어. </li></ol> 제 5 부 : Oocytes의 고정, 탈수 및 저장 <ol><li> 어떤 죽은 oocytes를 정렬 및 유리 튜브에서 살아남기 oocytes를 배치합니다. 우리는 일상적으로&gt; 90 % 생존을 관찰. </li><li> MEMFA 정착액 (재료 및 방법 참조) 20 분 바위 플레이스 oocytes. 알루미늄 호일로 덮고하여 빛으로부터 oocytes를 보호합니다. </li><li> 정착액을 제거하고 MBSH 버퍼의 동일한 볼륨 대체하여 oocytes 씻으십시오. 이 세척의 총 한 번 더이 세차를 반복합니다. </li><li> 무수 메탄올에 oocytes 씻으십시오 : <ol class="lalpha"><li> 볼륨의 절반을 제거 메탄올로 바꿉니다. </li><li> &quot;A&quot;두 단계를 반복합니다. </li><li> 솔루션의 모든 제거 메탄올로 바꿉니다. </li><li> 메탄올 세척을 반복합니다. </li></ol></li><li> Oocytes는 이미지를 준비하기 전까지 -20 ° C에 저장할 수 있습니다. </li></ol> 6 부 : 공촛점 현미경으로 영상 RNA의 현지화 <ol><li> 이미징 전에 이미지 표면을 커버하기 위해 유리 바닥 FluoroDish (WPI 주)에 머리 S 지우기 매체를 (2시 1분 벤질 벤조 산 벤질 알코올 -) 추가합니다. </li><li> 조심스럽게 양도 메탄올의 볼륨을 최소화, 메탄올에서 FluoroDish로 oocytes을 전송하기만하면됩니다. </li><li> 거꾸로 공촛점 현미경에 이미지 (우리는 성공적으로 자이스 혈구 LSM510와 Leica TCS SP2를 사용했습니다.) </li></ol><img src="/files/ftp_upload/1704/1704fig1.jpg" alt="그림 1" /> 그림 1 :.. 찬란 Labelled 성적 Microinjection하여 Subcellular RNA 현지화의 시각화 알렉사 플루어 546으로 표시 A. 따라 microinjection, RNA가 처음에 핵에 제한됩니다 B.이 문화 여덟 시간 후, 찬란 표시 제어 RNA가 균일하게 볼 수 있습니다 oocyte의 세포질에서 배포. C. 그러나, 수송 기계를 모집 해당 시퀀스를 포함하는 찬란 표시된 RNA가 oocyte (아래 방향)의 식물 극에 subcellular 지방화의 과정에서 볼 수 있습니다. 스케일 바 = 50 μm의.

<ol>
	<li>Cohen, S., Au, S., Pant, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Xenopus+laevis+oocytes.">Microinjection of Xenopus laevis oocytes.</a> JoVE. 24, (2009).</li><li>Dumont, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis+in+Xenopus+laevis+(Daudin).+I.+Stages+of+oocyte+development+in+laboratory+maintained+animals.">Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals.</a> J. Morphol. 136, 153-179 (1972).</li><li>Gagnon, J. A., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+transport+in+ovo:+Simultaneous+visualization+of+two+RNAs.">RNA transport in ovo: Simultaneous visualization of two RNAs.</a> Mol Reprod Dev. 76, 1115-1115 (2009).</li><li>Gautreau, D., Cote, C. A., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+copies+of+a+subelement+from+the+Vg1+RNA+localization+sequence+are+sufficient+to+direct+vegetal+localization+in+Xenopus+oocytes.">Two copies of a subelement from the Vg1 RNA localization sequence are sufficient to direct vegetal localization in Xenopus oocytes.</a> Development. 124, 5013-5020 (1997).</li><li>Lewis, R. A., Gagnon, J. A., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTB/hnRNP+I+is+required+for+RNP+remodeling+during+RNA+localization+in+Xenopus+oocytes.">PTB/hnRNP I is required for RNP remodeling during RNA localization in Xenopus oocytes.</a> Mol Cell Biol. 28, 678-6786 (2008).</li><li>Messitt, T. J., Gagnon, J. A., Kreiling, J. A., Pratt, C. A., Yoon, Y. J., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+kinesin+motors+coordinate+cytoplasmic+RNA+transport+on+a+subpopulation+of+microtubules+in+Xenopus+oocytes.">Multiple kinesin motors coordinate cytoplasmic RNA transport on a subpopulation of microtubules in Xenopus oocytes.</a> Dev Cell. 15, 426-436 (2008).</li><li>Mowry, K. L., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vegetal+messenger+RNA+localization+directed+by+a+340-nt+RNA+sequence+element+in+Xenopus+oocytes.">Vegetal messenger RNA localization directed by a 340-nt RNA sequence element in Xenopus oocytes.</a> Science. 255, 991-994 (1992).</li><li>Yoon, Y. J., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+Staufen+is+a+component+of+a+ribonucleoprotein+complex+containing+Vg1+RNA+and+kinesin.">Xenopus Staufen is a component of a ribonucleoprotein complex containing Vg1 RNA and kinesin.</a> Development. 131, 3035-3045 (2004).</li></ol>

실험 동물을 사용하는 실험은 브라운 대학에서 정한 지침과 규정에 따라 수행되었다.

여기 우리는 Xenopus의 oocytes에서 mRNA의 현지 시각을위한 프로토콜을 제시했습니다. 이 방법은, 찬란 표시 RNA의 성적표를 사용하면 이전에 dig​​oxigenin - 표시된 성적표와 함께 취득하고 간단하고 원위치 기반 접근법 (마우리과 멜튼, 1992, 고터오 외., 1997)보다 빠르고보다 노이즈 비율 높은 신호를했습니다. 이 방법을 사용하여, 우리는 엔지니어 순서 변이를 RNA 빠르게 생체내 기능에 ?...

A correction to the step 2 of Part 1 for article Visualizing RNA Localization in Xenopus Oocytes.

Erratum: Visualizing RNA Localization in Xenopus Oocytes

10X 텍사스 버퍼 <ul><li class="none"> 60 MM MgCl 2 </li><li class="none"> 400 MM 트리스 - HCL (산도 7.5) </li><li class="none"> 20 MM spermidine - HCL </li></ul> 20x 모자 / NTP 믹스 <ul><li class="none"> 10 MM CTP </li><li class="none"> 10 MM ATP </li><li class="none"> 9 밀리 UTP </li><li class="none"> 이 MM GTP </li><li class="none"> 20 MM G (PPP) G 총액 아날로그 (뉴 잉글랜드 Biolabs) </li></ul> G - 50 컬럼 <ul><li class="none"> 100 ML의 수화물 5g Sephadex G - 50 비즈 (시그마 알드리치) H 2 O.을 deionized 30 분 DEPC는 - 치료. 그리고 압력솥. 실온에서 불완전한 주식을 저장합니다. 사용하기 전에, 다음 RNase 무료 솔루션을 추가합니다 : </li><li class="none"> 0.5 ML 0.2 M EDTA (에틸렌 다이아 민 테트라 초산) </li><li class="none"> 1 ML 1 M 트리스의 산도 8.0 </li><li class="none"> 0.5 ML 20% SDS </li><li class="none"> 4 ° C.에 완전한 G - 50 솔루션을 저장 </li><li class="none"> 제거하고 폐기 플런저를 3 ML 주사기 (BD Biosciences)로부터 15 ML 원뿔 튜브 (코닝)에 주사기의 총구를 놓습니다. 글라스울 (동전의 절반 크기 플러그)의 소량으로 주사기를 꽂습니다. </li><li class="none"> 구슬을 resuspend하는 소용돌이 완료 G - 50 솔루션입니다. </li><li class="none"> 빈 열 2 ML G - 50 솔루션을 추가합니다. </li><li class="none"> benchtop의 원심 분리기 1,000 X g에서 1 분간 돌린다. </li><li class="none"> 각 열의 200 μl DEPC - 처리 deionized H 2 O를 추가합니다. 스핀. </li><li class="none"> 세 세척 총 두 번 더 씻어 반복합니다. </li><li class="none"> 새로운 15 ML 원뿔 관에 주사기 배럴을 제거합니다. </li></ul> Collagenase 솔루션 <ul><li class="none"> 클로스 트리 디움 histolyticum (시그마 알드리치)에서 75 MG collagenase </li><li class="none"> 25 ML 0.1 M KPO 3 + (산도 7.4) </li></ul> MBSH 버퍼 <ul><li class="none"> 88 MM NaCl </li><li class="none"> 1 ㎜ KCl </li><li class="none"> 2.4 MM NaHCO 3 </li><li class="none"> 0.82 MM MgSO 4 7H 2 O X </li><li class="none"> 0.33 MM 칼슘 (NO 3) 2 X 4H 2 O </li><li class="none"> 0.41 MM CaCl 2 X 6H 2 O </li><li class="none"> 10 MM HEPES (산도 7.6) </li></ul> Oocyte 문화 미디엄 <ul><li class="none"> 50% L15 미디어 </li><li class="none"> 15 MM HEPES (산도 7.6) </li><li class="none"> 1 MG / ML 인슐린 </li><li class="none"> 100 MG / ML gentamicin </li><li class="none"> 50 U / ML nystatin </li><li class="none"> 50 U / ML 페니실린 </li><li class="none"> 50 MG / ML 스트렙토 마이신 </li></ul> MEMFA 솔루션 <ul><li class="none"> 0.1 M의 대걸레 (산도 7.4) </li><li class="none"> 이 MM EGTA </li><li class="none"> 1 ㎜ MgSO 4 </li><li class="none"> 3.7 %의 포름 알데히드 </li></ul> 컴퓨팅 RNA 수율 <ul><li class="none"> &quot;입력&quot;과 표준 신틸레이션 계수기를 사용하여 &quot;통합&quot;샘플의 노출당 비용 (CPM)를 결정합니다. </li><li class="none"> 법인 = ( &quot;법인&quot;) / (10 X &quot;입력&quot;) </li><li class="none"> 일반 정관 값은 0.03 ~ 0.10 사이의 범위와. </li><li class="none"> 다른 polymerases에 대한 최대 산출 이론 : T3 T7, SP6 - 2.64 μg </li><li class="none"> μg = (효소의 최대 수율이 사용) X (정관)의 반응 수율 </li><li class="none"> 50 NM = (μg RNA) /로 RNA를 희석 320 / (기지에서 RNA의 길이) / (5X10 -8) </li><li class="none"> 반응은 일반적으로 수확량 ~ 50 NM에서 RNA의 50-100 μl. </li></ul>

visualizing rna localization in xenopus oocytes

RNA의 지방화는 세포 극성을 설립의 보존 메커니즘입니다. Vg1 mRNA는 Xenopus laevis oocytes과 공간의 Vg1 단백질의 유전자 발현을 제한하는 행위의 식물 극을 localizes. 이러한 방식으로 Vg1 배포 꽉 제어는 개발 배아에서 적절한 세균 계층 사양이 필요합니다. mRNA의 3 &#39;UTR에서 RNA의 시퀀스 요소는 Vg1 지역화 요소 (VLE)가 필요하며 직접 전송하기에 충분합니다. 생체내에 Vg1 mRNA의 인식 및 전송을 연구하기 위해, 우리는 단순한 시각적인 판독을 통해 트랜스 팩터 감독 전송 메커니즘의 광범위한 분석을 수있는 이미징 기술을 개발했습니다. RNA의 지방화를 시각화하기 위해, 우리는 찬란 표시 VLE의 RNA를 합성하고 개별 oocytes에 대한 본 사항이 명시를 microinject. 주입된 RNA의 전송을 허용하는 oocyte 문화 후, oocytes 사전 공촛점 현미경으로 영상을 고정하고 탈수 있습니다. mRNA의 현지화 패턴의 시각화는 RNA 수송의 전체 경로를 모니터링 및 CIS - 행동 VLE (루이스 외., 2008에 바인딩 증명서 및 횡단 행동 요소 내의 요소에 대한 RNA 수송 감독의 역할을 식별에 대한 기록을 제공합니다 Messitt 외., 2008). 우리는 추가 RNAS과 단백질 (가뇽과 마우리, 2009 Messitt 외., 2008)와 함께 공동 지방화를 통해이 기술을 확장 있고, 모터 단백질의 파괴와 cytoskeleton와 함께 (Messitt 외., 2008) 탐침을 mRNA의 지방화를 기본 메커니즘.

Watch this Scientific Journal Video about Visualizing RNA Localization in Xenopus Oocytes at JoVE.com

Visualizing RNA Localization in Xenopus Oocytes

의 시각화<em&gt; 생체내에</em&gt; RNA 교통이 찬란 표시 RNA의 성적의 microinjection에 의해로 수행됩니다<em&gt; Xenopus</em&gt; oocytes, 공촛점 현미경으로 따라갔다.

머릿속 RNA 현지화에서 Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

visualizing-rna-localization-in-xenopus-oocytes

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Visualizing RNA Localization in Xenopus Oocytes

11.5K 조회수.  Brown University. 의 시각화 생체내에 RNA 교통이 찬란 표시 RNA의 성적의 microinjection에 의해로 수행됩니다 Xenopus oocytes, 공촛점 현미경으로 따라갔다.

비디오: Visualizing RNA Localization in Xenopus Oocytes

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Xenopus Oocytes의 표현 이온 채널 패치 클램프 녹음

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

연구

생물학

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 ...

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 ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

이온 채널 유학을위한 패치 클램프 및 재관류 기법으로 표현 Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

HELA 세포 Transfection 얹는 Gaussia의 루시페라제의 mRNA의 시험 관내 스크립트 작성 및 상한에

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

오세영, K와 H, K-ATPase에 의해 양이온 교통 측정<em&gt; Xenopus</em원자 흡수 분광 광도법에 의한&gt; Oocytes : 방사성 동위 원소의 Assays에 대한 대안

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

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. ...

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery (References 2 - 3, and references therein). A characteristic that makes the oocyte attractive for foreign channel expression is the poor abundance of endogenous ion channels4. This expression system has proven useful for the characterization of many proteins, among them ligand-gated ion channels.
The expression of GABAA receptors in Xenopus oocytes and their functional characterization is described here, including the isolation of oocytes, microinjections with cRNA, the removal of follicular cell layers, and fast solution changes in electrophysiological experiments. The procedures were optimized in this laboratory5,6 and deviate from the ones routinely used7-9. Traditionally, denuded oocytes are prepared with a prolonged collagenase treatment of ovary lobes at RT, and these denuded oocytes are microinjected with mRNA. Using the optimized methods, diverse membrane proteins have been expressed and studied with this system, such as recombinant GABAA receptors10-12, human recombinant chloride channels13, Trypanosome potassium channels14, and a myo-inositol transporter15, 16.
The methods detailed here may be applied to the expression of any protein of choice in Xenopus oocytes, and the rapid solution change can be used to study other ligand-gated ion channels.

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented.

Xenopus의 난 모세포 : 미세 주입을위한 최적화 방법, 여포 세포 레이어의 제거 및 전기 생리 실험에서 빠른 해결 방법 변경

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

TChIP-Seq: 셀 형식 관련 Epigenome 프로 파일링

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.