Name: xenopus laevis as a model to identify translation impairment
Uploaded: 2015-09-27T14:00:00
Description: Watch this Scientific Journal Video about Xenopus laevis as a Model to Identify Translation Impairment at JoVE.com

This research was supported by grant from INSERM, University of Lille 1, University of Lille 2, Regional Hospital Center of Lille (CHR de Lille). MCCH acknowledges supports from the Fondation de France and wishes to thank IRCL, Pr. Sonenberg for the gift of the V5-plasmids, Dr. Dissous (Pasteur Institute, Lille) for the gift of anti-GFP antibodies, UMS 3387 (University of Rennes) where Xenopus oocytes are purchased and Dr Taymans (JPArc, Lille) for critical reading of the manuscript text.

모든 Xenopus의 실험은 실험실 동물 실험에 대한 유럽 공동체위원회 지침 (609분의 86 / EEC)의 규칙에 따라 릴 1 대학의 동물 시설에서 수행 하였다. 동물 프로토콜 (위원회 (CCT) 디부 Ethique 실내 실험 ANIMALE 노르 파 드 칼레 CEEA 07/2010)을 지역 기관 심사위원회에 의해 승인되었다. 1. 난자 처리 <ol><li> 마취 솔루션을 준비 : 멸균 물 1 L에 tricaine 메탄 설포 네이트 분말 1g을 용해...

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번역은 여러 가지 신경 퇴행성 질환 등 다양한 인간 질환의 physiopathology에 관여하는 메커니즘입니다. 파킨슨 병의 예를 들어 여러 보고서 유전 변이 8,12,13과 관련된 번역에 손상을 제안했다. 여러 셀룰러 모델은 번역을 연구하는 데 사용할 수 있습니다. 여기서, eIF4G1 및 8의 eIF4E 파트너 간의 상호 작용을 감소시키는 등의 우성 네가티브 돌연변이 작용 eIF4G1에?...

단백질은 생물체의 큰 규모에 필수적인 세포 삶의 요소 때문에입니다. 이들은 구조, 운송, 반응 촉매, 조절, 유전자 발현 등 발현은 mRNA의 단백질로의 변환을 허용하는 번역 복잡한 메커니즘의 결과를 포함한 세포 기능의 대부분을 보장한다. 번역은 적응 및 발생과 분화, 노화, 스트레스 또는 생리 병리학 적 양상 동안, 셀의 요구에 따라 유전자 발현을 조절하는 각종 제어를 실시한다. 캡에 의존, 모자 독립적 인 내부 리보솜 항목 세그먼트 (IRES) 구조와 캡 독립 번역 약물 (경유 : 번역은 3 개시 번역 시스템이 요구에 대응하기 위해 3 단계 (개시, 신장 및 종료)와 선물로 분할된다 ) CITE. 대부분의 진핵 생물의 mRNA는 캡 depe로 번역된다단백질 합성시 인식 기능의 역할 7 methylguanosine 5&#39;- 삼인산 캡을 통해 ndent 방식. 이 모자의 eIF4E, eIF4G1 및 eIF4A와 eIF4F 단지의 구성 요소에 바인딩합니다. 폴리 (A) 결합 단백질 (PABP)와 같은 다른 파트너와 관련, eIF2-GTP-메트로-tRNA의 메트로,이 번역 개시 인자의 mRNA를 회람 및 인식 1 코돈 8 월 개시 될 때까지 형태 43S 복잡한 접근성을 개선 할 수 있습니다. 이 이벤트는 번역 개시 즉, 변환의 제 1 단계의 종료에 대응한다. 캡 독립적 번역 인스턴스 세포 증식 및 아폽토시스 유도에 대한 스트레스 조건 하에서 필수적인 단백질 mRNA의 인코딩에 의해 사용된다. 이 메커니즘은 mRNA의에 차 구조를 포함 5&#39; 번역되지 않은 지역 (UTR)는 IRES라고 eIF4A 및 43S 복합과 관련된 eIF4G1의 카르복시 말단. 이 43S 개시전 (C)의 결합IRES에 omplex은 eIF4E를 인자 2,3 필요없이 뚜껑 독립적 인 번역을 시작합니다. mRNA의 UTR 4 내에있는 구조를 인용을 통해 마지막으로, 아직 잘 이해되지 다른 변환 메커니즘은 스트레스 조건 하에서이 모자 독립적 인 번역 활동을 지원합니다. 그들의 개시 단계에서 다른 번역이 다양한 방식을 통해 변환이 셀룰러 항상성에 중요한 역할을하고, 따라서 큰 규모의 효과와 작은 유기체에 영향을 미칠 것이다 이러한 프로세스 중 하나에 변경을 수행한다. 실제로, 개시는 mRNA의 단백질로의 정확한 변환을 관리하는 프로세스 속도 제한 단계이며, 따라서 다수의 제어 및 조절 점 (5)의 목표이다. 이 후자의 또는 이러한 프로세스의 구성 요소에 대한 여부 하나에 결함이있는 것으로 밝혀지면, 그것은 셀의 설립 균형을 교란하고, 따라서 병리 콘돌리자으로 이어질 수TIONS. 이러한 맥락에서, 번역 인자의 돌연변이는 이러한 잠재적 흰색 matter&#39; (eIF2B1-5 서브 유닛) (6), 월콧 - Rallison 증후군 (PERK에 대한 EIF2AK3 유전자 인코딩) (7), 소멸과 백색질 뇌증 &#39;와 같은 퇴행성 신경 질환 등 여러 질환에 참여하고있다 파킨슨 병 (eIF4G1의 p.R1205H) 8. 이는 질병 개발 및 번역 개시의 일반적인 과정에 대한 우리의 지식을 증가시키는 이러한 돌연변이 단백질의 세포 및 분자 연구를 수행하는 것이 중요하다. 이 연구를 수행하기 위해, 이러한 돌연변이의 영향을 관찰하기 위해 가장 적절한 모델을 선택하는 것이 필수적이다 제노는 난자 특히 생리 학적 및 생화학 적 특성으로 인해 적응 laevis의 :. 생리 동시성 (세포주기의 단계 G2에서 차단) , 단백질 합성의 높은 용량 (200 ~ 400 NG / 일 / 난자), 추출 OOC의 높은 수같은 동물에서 ytes (800-1,000 난자 / 여성)과 조작을 용이하게 셀 크기 (직경 1.2-1.4 mm)이다. 합성의 mRNA와 제노 푸스 난 모세포의 마이크로 인젝션 쉽게 번역 단계를 해부하기 위해 수행 될 수있다. 이보기에 그것은 다른 장점을 제공합니다. 감수 분열의 진행의 속도와 번역의 mRNA의 미세 주입 (~ 24 시간) 후 감안할 때, Xenopus의 난자는 재구성 세포 (대장균에서 추출, 밀 세균 또는 토끼 망상 ...) 시스템의 mRNA가 인에 비해 빠른 시스템을 나타냅니다 감소 된 변환 속도와 낮은 속도로 번역했다. 따라서 mRNA의 도입으로 돌연변이의 효과를 신속하고 용이하게 관찰 할 수있는 여러 개의 난 모세포에서 연구 될 것이다. 제노 푸스 난 모세포의 또 다른 장점은 모체의 mRNA가 잠재되어 단백질 번역을 프로게스테론 자극 전에 차단된다는 것이다. 프로게스테론 첨가 따라서 변환 유도를 제어하는​​ 좋은 방법이다. 세포질 Polyadenylation는 oogenesis 동안 발생하지 않습니다. 그것은 시간적 순서에 프로게스테론 자극 난자에 난자 성숙 과정 시작과 초기 개발을 통해 계속 번역의 다른 단계를 연구하는 데 사용할 수 있습니다. MOS의 mRNA의 폴리아 데 닐화가 발생하는 첫 번째 중 하나입니다 그것은 찰스 워드 등에 정의 된 &quot;조기 성숙&quot;유전자의 클래스에 오로라 / EG2, 히스톤 같은 B4의 mRNA에 속한다. (2004) 9. 이러한 사이클린 A1과​​ B1 사이클린 등의 &quot;말&quot;의 mRNA의 번역 유도 새싹 소포 고장 (GVBD)의시기에 발생합니다. MOS의 mRNA는 세린 / 트레오닌 - 단백질 키나아제를 암호화한다. 이 간접적으로 난자의 성숙을 활성화 MAP 키나제 캐스케이드을 유도하기 때문에 그것의 번역은 매우 중요하다. 사실, 황체 호르몬에 대한 응답으로, MOS의 mRNA의 폴리아 데 닐화는 EG2 조절 단백질과 일 다른 RNA 결합 단백질을 오로라 /를 포함하는 과정을 통해 강화된다MOS의 mRNA의 전자 3&#39;UTR. MOS의 mRNA의 폴리아 데 닐화 증가 차례로 MEK1를 활성화 MOS 단백질 수준의 증가를 이끈다. 이 과정은 세포 외 신호 조절 키나아제 2 (ERK2) (도 1)의 활성화를 매개한다. 이 시그널링 캐스케이드 다음 요인을 촉진 성숙 M 상, 사이클린 B 및 Cdc2 키나제에 의해 형성된 복합체를 유발하며 결국 감수 재개 야기 할 수있다. 제노 푸스는 난자 laevis의 그러므로, 이러한 MOS 등 산모의 mRNA의 연구는 쉽게 여러 MOS 부품 시그널링도 GVBD 레이트의 판정 등의 번역 그들의 효율적인 폴리아 데 닐화에서 여러 엔드 포인트와 그 번역 가능성을 테스트하기 위해 사용될 수있다. 이 시스템은 신규의 mRNA의 전사 또는 형질 감염 효율의 간섭없이 번역 개시 인자의 돌연변이의 첫번째 결과를 평가하는 것이 흥미 롭다는 문제가 종종 함께 발생 eukary귀 세포 연구. 여기에, 프로토콜은 Xenopus의이 난자를 laevis의 산모의 mRNA의 번역 시험에서 돌연변이 eIF4G1의 mRNA를가 미세 주입되는 경우 설정됩니다. 난자 감수 세포주기를 통해 초기와 후기 클래스의 mRNA의 후속 번역 진행을 위해 필수적이다 GVBD 진행에 결함, MOS mRNA의 폴리아 데 닐화의 존재에서 확인된다. 인산화 오로라 / EG2와 ERK는 MOS의 deregulation.Thus의 결과를 확인하기 위해 연구되고, Xenopus의 난자는 mRNA의 번역의 다른 단계를 분석하는 간단한 방법을 나타냅니다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Tricaine methane sulphonate</td>
			<td>Sandoz</td>
			<td>MS-222</td>
			<td rowspan="8">oocytes handling</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Moria</td>
			<td>Dumont MC40</td>
		</tr>
		<tr>
			<td>Streptomycin/penicillin</td>
			<td>Sigma</td>
			<td>781</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma</td>
			<td>P2256</td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor</td>
			<td>Sigma</td>
			<td>T9128</td>
		</tr>
		<tr>
			<td>Tetracyclin</td>
			<td>Sigma</td>
			<td>T7660</td>
		</tr>
		<tr>
			<td>Veterinarian absorbable Vicryl thread</td>
			<td>Johnson&#38;Johson Intl</td>
			<td>JV1205</td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>Roche diagnostics</td>
			<td>10103586001</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PmeI</td>
			<td>New England Biolabs</td>
			<td>R0560S</td>
			<td rowspan="14">Preparation of synthetic mRNA</td>
		</tr>
		<tr>
			<td>DNAse/RNAse free H2O</td>
			<td>Life Technologies</td>
			<td>10977</td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Merck</td>
			<td>6268</td>
		</tr>
		<tr>
			<td>Absolute ethanol</td>
			<td>Sigma</td>
			<td>02854</td>
		</tr>
		<tr>
			<td>Nano Drop</td>
			<td>Thermo Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>TBE 10X</td>
			<td>Eppendorf</td>
			<td>0032006.507</td>
		</tr>
		<tr>
			<td>Ethidium bromide 10 mg/mL</td>
			<td>Life Technologies</td>
			<td>15585-011</td>
		</tr>
		<tr>
			<td>mMESSAGE mMACHINE&#174; Kit</td>
			<td>Ambion by Life Technologies</td>
			<td>AM1344</td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma</td>
			<td>M1254</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fluka</td>
			<td>03609</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Life Technologies</td>
			<td>16500-500</td>
		</tr>
		<tr>
			<td>Formaldehyde 37%</td>
			<td>Merck</td>
			<td>1.04003.1000</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Fluka</td>
			<td>47671</td>
		</tr>
		<tr>
			<td>Gel Doc Imager</td>
			<td>Biorad</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oocyte Pipet with 100 8&#34; Glass Capillaries</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-510-X</td>
			<td rowspan="4">microinjection</td>
		</tr>
		<tr>
			<td>Replacement Glass 8&#34;</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-210-G8</td>
		</tr>
		<tr>
			<td>0,45 &#181;m filter</td>
			<td>Millipore</td>
			<td>SLHA025NB</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P0130</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td rowspan="32">Western Blot</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S5886</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Biorad</td>
			<td>161-0301</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>A3294</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A4612</td>
		</tr>
		<tr>
			<td>leupeptin</td>
			<td>Sigma</td>
			<td>L8511</td>
		</tr>
		<tr>
			<td>aprotinin</td>
			<td>Sigma</td>
			<td>A1153</td>
		</tr>
		<tr>
			<td>benzamidine</td>
			<td>Sigma</td>
			<td>B6506</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma</td>
			<td>P7626</td>
		</tr>
		<tr>
			<td>sodium vanadate</td>
			<td>Sigma</td>
			<td>S6508</td>
		</tr>
		<tr>
			<td>Laemmli buffer</td>
			<td>Biorad</td>
			<td>161-0737</td>
		</tr>
		<tr>
			<td>NuPAGE Novex 4-12% Bis-Tris Protein Gels, 1.0 mm, 15 well</td>
			<td>Life Technologies</td>
			<td>NP0323BOX</td>
		</tr>
		<tr>
			<td>SDS-PAGE electrophoresis, mini-Protean TGX</td>
			<td>Biorad</td>
			<td>456-1036 and -1096</td>
		</tr>
		<tr>
			<td>horizontal semi-dry blotting system</td>
			<td>W.E.P. Compagny</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Biorad</td>
			<td>161-0718</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Biorad</td>
			<td>161-0719</td>
		</tr>
		<tr>
			<td>Hybond ECL Membrane</td>
			<td>Amersham Life Science</td>
			<td>10401180</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>VWR</td>
			<td>20846-292</td>
		</tr>
		<tr>
			<td>Ponceau Red (0,5%)</td>
			<td>Sigma</td>
			<td>P3504</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P2287</td>
		</tr>
		<tr>
			<td>anti-GFP</td>
			<td>Life Technologies</td>
			<td>A11122</td>
		</tr>
		<tr>
			<td>anti-V5</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-58052</td>
		</tr>
		<tr>
			<td>anti-Eg2</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-27884</td>
		</tr>
		<tr>
			<td>anti-Eg2-P</td>
			<td>Cell Signaling</td>
			<td>C39D8</td>
		</tr>
		<tr>
			<td>anti-ERK2</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-1647</td>
		</tr>
		<tr>
			<td>anti-ERK2-P (Tyr204)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-7976</td>
		</tr>
		<tr>
			<td>anti-Rsk</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-231</td>
		</tr>
		<tr>
			<td>anti-mos</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-86</td>
		</tr>
		<tr>
			<td>anti-mouse horseradish peroxidase labeled secondary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2005</td>
		</tr>
		<tr>
			<td>anti-rabbit horseradish peroxidase labeled secondary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2812</td>
		</tr>
		<tr>
			<td>anti-goat horseradish peroxidase labeled secondary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2378</td>
		</tr>
		<tr>
			<td>Advanced ECL Detection System</td>
			<td>Amershan Life Science</td>
			<td>RPN2135</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1x</td>
			<td>Sigma</td>
			<td>P4417</td>
			<td rowspan="11">polyadenylation assay</td>
		</tr>
		<tr>
			<td>TRIZOL (Qiazol Lysis Reagent)</td>
			<td>Qiagen</td>
			<td>79306</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma</td>
			<td>31998-8</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma</td>
			<td>278475-1L</td>
		</tr>
		<tr>
			<td>RNeasy mini kit</td>
			<td>Qiagen</td>
			<td>74106</td>
		</tr>
		<tr>
			<td>RTL buffer&#160;</td>
			<td>Qiagen</td>
			<td>79216</td>
		</tr>
		<tr>
			<td>RNAse free DNAse set</td>
			<td>Qiagen</td>
			<td>79254</td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>Eurogentec</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 RNA ligase 1&#160;</td>
			<td>New England Biolabs</td>
			<td>M0204S</td>
		</tr>
		<tr>
			<td>High capacity c-DNA Reverse Transcription kit</td>
			<td>Applied Biosystems, Life Technologies</td>
			<td>4368813</td>
		</tr>
		<tr>
			<td>Taq polymerase</td>
			<td>Life Technologies</td>
			<td>10342020</td>
		</tr>
	</tbody>
</table>

xenopus laevis as a model to identify translation impairment

단백질 합성은 다양한 생물학적 과정, 특히 환경 조건에 적응에 영향을 미치는 유전자 발현에 중요한 공정이다. mRNA의 개시 코돈, eIF4G1을 포함하여 참여 개시 인자의 리보솜 서브 유닛의 조립을 포함 개시 단계. 번역의 속도 제한 단계에서의 결함은 다양한 질환에 연결되어 있습니다. 이러한 규제 완화의 잠재적 결과를 연구하기 위해, 제노 푸스는 난자가 인간에 필수적인 세포 및 분자 메커니즘의 보존의 높은 학위를 가진 매력적인 모델을 구성 laevis의. 또한, 감수 성숙하는 동안, 난 모세포는 전사적으로 억압하고 필요한 모든 단백질은 기존의, 모계 유래의 mRNA에서 변환됩니다. 이 저렴한 모델은 효과적인 번역과 완벽하게 통합 될 외인성 mRNA를 할 수 있습니다. 여기에 (여기 eIF4G1) 관심 요인으로 번역을 평가 STOR 사용을위한 프로토콜을 설명하는가장 먼저 그 어머니의 mRNA의 에드 폴리아 데 닐화 및 생리 학적 판독으로 난자의 성숙하는 동안 번역합니다. 처음에는 mRNA의 관심의 플라스미드 (여기 eIF4G1)의 시험 관내 전사에 의해로 합성하는 것은 어린 싹 소포 고장 감지하여 난자와 난자의 성숙의 역학에 주입이 결정된다. 연구 모체 mRNA의 타깃은 세린 / 트레오닌 - 단백질 키나아제 MOS이다. 그 폴리아 데 닐화 및 그 이후의 번역은 난자의 성숙에 관여 폭포 신호 MOS의 단백질의 발현 및 인산화와 함께 조사하고 있습니다. 현재 프로토콜의 변화는 앞으로 넣어 번역 결함은 또한 일반적인 적용 성을 강조하기 위해 제안되었다. 비정상적인 단백질 합성은 신경 질환의 병인에 관여 할 수 있다는 증거를 부상에 비추어, 그러한 모델은 쉽게 손상을 평가하고, 새로운 대상을 식별 할 수있는 기회를 제공한다.

PG 자극의 24 시간 (도 2B, 2C) 후 Xenopus의 난자와 백분율 난자 GVBD 결정의 운동 성숙 : eIF4G1-DN 돌연변이 병진 영향을 연구하기 위해, 제노 푸스에서 PG에 대한 응답은 (H 2 O, GFP) eIF4G1-WT와 다른 제어 상태와 비교 된 cRNA eIF4G1-DN 마이크로 인젝션 된 난자 laevis의. 컨트롤에 관계없이 주입 된 cRNA 또는 eIF4G1 과발현의 성격의 난자 미세 주사의 발?...

Watch this Scientific Journal Video about Xenopus laevis as a Model to Identify Translation Impairment at JoVE.com

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 ...

xenopus-laevis-as-a-model-to-identify-translation-impairment

Research

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Biology

Xenopus laevis as a Model to Identify Translation Impairment

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

주최자 및 동물 폴 Explants Xenopus laevis의 태아로부터 세포 접착 분석의 총회의 해부

연구

생물학

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

플라스틱 삽입 및 Xenopus laevis의 태아의 Sectioning

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

Xenopus laevis 여성의 난자를 구하기

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Xenopus laevis 에그의 작성과 분류는 압축을 풉니다

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

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 ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

에있는 동물 캡 이식 (ACT) 어세를 사용하여 조직 결정 Xenopus laevis</em

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

라이브 셀 이미징 및 배아 상피 세포의 정량 분석 Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

생산<em&gt; Xenopus의 tropicalis</em&gt; 달걀은 미세 소관 관련 RNA를 식별하기 위해 추출

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

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. ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

에 미세 소관 역학을 측정하는 plusTipTracker 소프트웨어를 사용<em&gt; 운송에 필요한 정보 미국 교통부</em&gt; 성장 콘

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...