이 작품은 독일 Forschungsgemeinschaft (DFG)에 의해 지원되었다, 우리는 기술 지원을 Ayele Tsedeke Taddese을 감사드립니다.

1. 유리 바늘의 준비 (그림 1) <ol><li> 주사 바늘 (그림 1A)를 준비하기 위해 825 ㎜의 유리 모세관을 사용합니다. </li><li> 바늘 풀러에 유리 모세관을 놓고 다음과 같은 매개 변수를 사용하여 바늘을 당겨 : 난방 사이클 값 : 463; 사이클 값 당기는 : 230; 속도 : 150 밀리 초; 시간 : 150 밀리 초 (그림 1B). </li><li> 마이크로 미터의 접안 렌즈와 입체경에서 20 μm의 직경 바늘을 생산하는 시계 제조 핀셋 뽑아 유리 모세관 휴식. </li><li> 바늘을 날카롭게하고 20 μm의 경사 (그림 1C 및 1D)를 생산하기 위해 젖은 고무 물레와 선반을 사용합니다. 참고 : 샤프 빗면 바늘 뇌실에 삽입의 용이성을 향상시키고, 따라서 조직에 손상을 최소화하고 천공 근육 바늘 (도 1E-G)의 제거 후에 다시 봉인하는 것을 허용한다. </li></ol> 2. Prepar모르 폴리 솔루션의 ATION <ol><li> 7.5 mM의 최종 농도로 1X 인산 완충 생리 식염수 (PBS)에서 동결 건조 된 모르 폴리 용해시켜 모르 폴리 콘텐츠를 준비한다. [자세한 내용은 제조업체의 지침 (재료 표)를 참조]. </li><li> 3.5 MM의 모르 폴리 노 0.5 mM의 엔도 - 포터의 최종 농도 2.8 ㎕의 엔도 - 포터 원액 (1 ㎜) (재료 표 참조) 2.5 μL의 모르 폴리 노 원액 (7.5 mm)를 혼합하여 모르 폴리 노 주입 솔루션을 준비합니다. </li></ol> 3. 모르 폴리 노 주입의 준비 (그림 2) <ol><li> 10 μL microloader 피펫 팁과 마이크로 피펫을 사용하여이 솔루션의 5 μL와 경 사진 유리 바늘을 넣습니다. </li><li> 압축 공기를 넣은 피코 펌프 (그림 2A-C)에 연결된 미세 조작기의 바늘 홀더에 유리 바늘을 삽입합니다. </li><li> 다음 현미경으로 바늘 홀더를 놓고 바늘 만 요구되도록유충 (그림 2A)의 심실에 삽입 한 평면 방향으로 이동합니다. </li><li> 약 45 °에서 주사의 각도를 조정합니다. </li><li> 대기 압력 : 다음과 같이 microinjector 값을 설정 평방 인치 (PSI) 당 20 파운드; 토출 압력 : 15 PSI; 게이트의 100 밀리 초 범위는 1.9의 기간 값 (10.9 밀리 초에 해당). </li><li> 1.5 %의 20 ㎖ (w / v) 겔을 생산하는 표준 마이크로 웨이브를 이용하여 1X PBS에서 융해. 10cm 페트리 접시에 녹은 젤을 붓는다. 일단 경화, 아가로 오스가 진정 유충 (11) (그림 2D 및 2E)을 배치 할 수있는 고랑이되도록 따뜻한 아가로 오스에 홈이 분사 형태를 놓습니다. </li></ol> 4. 주사 (그림 3) <ol><li> 20 ㎎ / L의 tricaine (L-에틸-m-아미노 벤조 에이트 메탄 설포 네이트)가 터치 응답을 중지 할 때까지 수족관 물 100 ㎖에 애벌레를 마취. </li><li> 플라스틱 파스퇴르 pipett를 사용복부 측면은 홈 (그림 3A)의 수직 아가 벽에 향하도록 전자 신중하게 젖은 아가로 오스 금형의 홈에 진정 유충을 전송합니다. </li><li> 심실 멀리 주사 바늘 (그림 3A)에서 향하도록 실체 현미경 아가로 오스 플레이트를 놓습니다. </li><li> 심장 심실에 삽입 바늘을 낮 춥니 다. 만 1 ~ 2 μm의 너무 깊이 바늘 (그림 3B 및 3C)를 삽입되지 않도록주의 심실에 바늘을 삽입합니다. </li><li> 바늘이 삽입되면, 하트 (도 3D 및 3E)의 소거를 허용하도록 간격을 기다리고, 4-6 펄스, 용액의 각 전달 3 NL과 심실로 모르 폴리 용액을 주입. </li><li> 주사 후 바늘 (삽입의 평면에 평행)를 제거하고 조심스럽게 E3 매체의 혈중 알코올 농도로 채워진 플라스틱 파스퇴르 피펫을 사용하여 유충을 전송E3 신선한 배지를 포함하는 페트리 접시로 케이. </li><li> 주입 된 유충을 전송할 때 바늘의 건조를 막기 위해, 1X PBS를 함유하는 페트리 접시로 바늘을 배치. </li><li> 반복 4.1-4.6에게 실험 기간 동안 매일 12 ~ 24 시간을 반복합니다. 참고 : 주사를 반복하는 세포의 모르 폴리 노의 흡수 및 유지 보수를 보장합니다. </li></ol> 5. 주사의 분석 (그림 4) <ol><li> 그들은 터치 응답을 중지 할 때까지 20 ㎎ / L의 tricaine과 수족관 물 100 ㎖에 물고기를 마취. </li><li> 평면 젖은 1.5 %에 옆으로 유충을 배치 E3 매체로 덮여 (w / v) 아가로 오스 플레이트. </li><li> 시야 및 형광 이미징과 실체 현미경을 사용하여 이미지의 애벌레. 참고 : 유충이 투명으로, 플루 오레 태그 모르 폴리 노를 직접 주사 후 동물에 걸쳐 혈관에 형광 녹색으로 표시되어야합니다. 이 형광 추가하여 혈관 조직으로 분산됩니다15 분 (도 4A-4C). </li></ol> 재생 파생물의 6. 평가 <ol><li> 피지 이미지 J 무료 소프트웨어 (http://fiji.sc/Fiji)를 사용하여 촬영 한 이미지를 평가합니다. 재생을 조사하는, 제어 및 모르 폴리 주입 유충에 발생한 회생 성장의 양을 결정하기 위해 라인 추적 툴을 사용한다. </li><li> 이미지를 보정하기 위해, 끌어 주 피지 창에 파일을 드롭하여 피지에있는 원본 이미지 파일 중 하나를 엽니 다. </li><li> 다음의 모든 이미지의 배율을 설정하려면 주 메뉴에서 &quot;분석&quot;로 이동합니다. </li><li> &quot;설정 규모&quot;에 대한 메뉴를 클릭 드롭 다운. </li><li> 이 창에서 체크 박스를 클릭하여 &quot;글로벌&quot;옵션을 설정합니다. </li><li> 이제 드래그 앤 드롭 (단계 6.1 참조)에 의해 다시 회생 성장의 양을 측정하기 위해 화상을 연다. </li><li> 도구 모음에서 &quot;프리 핸드 선택&quot;도구를 선택합니다. </li><li> 측정 할 영역 (FI 둘러싸gures 4J-4L). </li><li> Ctrl 키를 누르면 + M. 이 정사각형 픽셀에 둘러싸인 영역의 값을 보여주는 새로운 &quot;결과&quot;창을 엽니 다. </li></ol>

<ol>
	<li>Gemberlin, M., Bailey, T. J., Hyde, D. R., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+for+complex+tissue+regeneration.">The zebrafish as a model for complex tissue regeneration.</a> Trends in Genetics. 29, 611-620 (2013).</li><li>Suster, M. L., Abe, G., Schouw, A., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon-mediated+BAC+transgenesis+in+zebrafish.">Transposon-mediated BAC transgenesis in zebrafish.</a> Nature Protocols. 6, 2011 (1998).</li><li>Skromne, I., Prince, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+perspectives+in+zebrafish+reverse+genetics:+moving+forward.">Current perspectives in zebrafish reverse genetics: moving forward.</a> Developmental Dynamics. 237, 861-882 (2008).</li><li>Zu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TALEN-mediated+precise+genome+modification+by+homologous+recombination+in+zebrafish.">TALEN-mediated precise genome modification by homologous recombination in zebrafish.</a> Nature Protocols. 10, 329-332 (2013).</li><li>Bedell, V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+genome+editing+using+a+high-efficiency+TALEN+system.">In vivo genome editing using a high-efficiency TALEN system.</a> Nature. 491, 114-118 (2012).</li><li>Kizil, C., IItzsche, A., Kaslin, J., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromanipulation+of+gene+expression+in+the+adult+zebrafish+brain+using+cerebroventricular+microinjection+of+morpholino+oligonucleotides.">Micromanipulation of gene expression in the adult zebrafish brain using cerebroventricular microinjection of morpholino oligonucleotides.</a> J Vis Exp. , (2013).</li><li>Hyde, D., Godwin, A., Thummel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+of+morpholinos+into+the+regenerating+adult+zebrafish+tail+fin.">In vivo electroporation of morpholinos into the regenerating adult zebrafish tail fin.</a> J Vis Exp. , (2012).</li><li>Thummel, R., Bailey, T., Hyde, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+of+morpholinos+into+the+adult+zebrafish+retina.">In vivo electroporation of morpholinos into the adult zebrafish retina.</a> J Vis Exp. , (2011).</li><li>Rieger, S., Kulkarni, R. P., Darcy, D., Fraser, S. E., K&#246;ster, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantum+dots+are+powerful+multipurose+vital+labeling+agents+in+zebrafish+embryos.">Quantum dots are powerful multipurose vital labeling agents in zebrafish embryos.</a> Developmental Dynamics. 234, 670-681 (2005).</li><li>Madhavan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+Pax-6+in+lens+regeneration.">The role of Pax-6 in lens regeneration.</a> Proceedings of the National Academy of Science U. 103, 14848-14853 (2006).</li><li>Gilmour, D. T., Jessen, J. R., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish: a practical approach. 261, 121-143 (2000).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9, 676-682 (2012).</li><li>Eisen, J. S., Smith, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+morpholino+experiments:+don't+stop+making+antisense.">Controlling morpholino experiments: don't stop making antisense.</a> Development. 135, 1735-1743 (2008).</li><li>Bill, B. R., Petzold, A. M., Clark, K. J., Schimmenti, L. A., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primer+for+morpholino+use+in+zebrafish.">A primer for morpholino use in zebrafish.</a> Zebrafish. 6, 69-77 (2009).</li><li>Hyatt, T. M., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vectors+and+techniques+for+ectopic+gene+expression+in+zebrafish.">Vectors and techniques for ectopic gene expression in zebrafish.</a> Methods Cell Biol. 59, 117-126 (1999).</li><li>Hans, S., Kaslin, J., Freudenreich, D., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally+controlled+site-specific+recombination+in+zebrafish.">Temporally controlled site-specific recombination in zebrafish.</a> PLoS One. 4, (2009).</li><li>Lee, Y., Grill, S., Sanchez, A., Murphy-Ryan, M., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf+signaling+instructs+position-dependent+growth+rate+during+zebrafish+fin+regeneration.">Fgf signaling instructs position-dependent growth rate during zebrafish fin regeneration.</a> Development. 132, 5173-5183 (2005).</li><li>Liu, S., D, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+models+for+cancer.">Zebrafish models for cancer.</a> Annu. Re. Pathol. Mech. Dis. 6, 71-93 (2011).</li><li>Stoick-Cooper, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+Wnt+signaling+pathways+have+opposing+roles+in+appendage+regeneration.">Distinct Wnt signaling pathways have opposing roles in appendage regeneration.</a> Development. 134, 479-489 (2007).</li><li>Shen, S. P., Aleksic, J., Russell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+targets+of+the+Sox+domain+proteins+Dichaete+in+the+Drosophila+CNS+via+targeted+expression+of+dominant+negative+proteins.">Identifying targets of the Sox domain proteins Dichaete in the Drosophila CNS via targeted expression of dominant negative proteins.</a> BMC Dev. Biol. 13, (2013).</li><li>McNeish, J. D., Scott, W. J., Potter Jr, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Legless,+a+novel+mutation+found+in+PHT1-1+transgenic+mice.">Legless, a novel mutation found in PHT1-1 transgenic mice.</a> Science. 241, 837-839 (1988).</li><li>Covarrubias, L., Nishida, Y., Terao, M., D'Eustachio, P., Mintz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+DNA+rearrangements+and+early+developmental+arrest+caused+by+DNA+insertion+in+transgenic+mouse+embryos.">Cellular DNA rearrangements and early developmental arrest caused by DNA insertion in transgenic mouse embryos.</a> Mol. Cell. Biol. 7, 2243-2247 (1987).</li></ol>

저자가 공개하는 게 없다.

뇌 실내 주입은 배아 발생 동안 유전자 기능에 영향을주지 않고 전개 또는 신체 항상성의 나중 단계에서 유전자 기능을 테스트하기위한 신속하고 신뢰성있는 평가 방법을 제공한다. 이 기술의 성공을 보장하기 위해, 하나는 네 가지 중요한 점을 알고 있어야합니다 바늘의 중 1) 바늘 크기, 2) 건조, 3) 양을 최소화하고, 진정 솔루션 4) 최소한의 노출 시간. 너무 큰 바늘 심실 손상과 과다 출혈의 원?...

장기와 부속의 재생은 생존과 피트니스에 대한 근본적으로 중요하다 그러나, 사람 등 여러 가지 척추 동물은 재생 능력을 제한했다. 여러 동물 모델은 광범위한 재생 능력을 가지고 존재하지만, 기관 및 부속 기관 재생하는 동안 유전자 기능을 평가하기 위해 유전자 기술을 반전은 매우 제한적이거나 존재하지 않는 남아 있습니다. 따라서 새로운 접근 방식은 이러한 모델 생물에서 중생의 분자 생물학을 해부하는 데 필요합니다. 제브라 피쉬는 여러 유전자 ​​변형 물고기 라인이 세포를 추적하기 위해 존재하기 때문에 셀 때문에 중요한 여러 장기, 조직 및 부속을 다시 생성 할 수있는 능력뿐만 아니라,의뿐만 아니라 기관 및 부속 기관 재생 1, 분자 생물학을 이해하기위한 기초가 튼튼한 모델입니다 유전자를 과발현하는 것은 2, 3를 구성한다. 그러나, 유생 지브라 피쉬의 유전자 억제는 주로 overexpre에 한정누구의 유전자 제품 유전자의 내인성 활성을 반영하지 않습니다 이득의 기능 효과를 얻을 수있는 모든 관심의 유전자 또는 사용할 수없는 지배적 인 음성 구조의 (투약) 소지. 따라서, 구체적으로 녹아웃 또는 넉다운하여 유전자 발현을 제거하는 다른 방법은 이러한 문제를 극복하기 위해 필요하다. TALENS은 유전자 기능을 녹아웃 역방향 유전자 수단으로서 존재하여 표적 유전자 특이; 유전자의 초기 요구 사항이 배아 발달의 추가 진행을 방지하기 때문에,이 녹아웃 전략은 매우 자주 초기 배 발생 중에 기능 평가로 제한됩니다. 따라서, 이러한 TALENS를 사용하여 개발 한 후 재생 또는 기관의 항상성 등의 이상 현상을 연구하는 5 4를 배제한다. 따라서 대체 유전자 제거 전략은 완전히 형성 장기 및 구조 유전자 요건을 평가하는 초기 현상 후 유전자 기능을 대상으로하는 필요가있다. </P&gt; 모르 폴리 노를 주사 몇 성인 장기에 유전자를 대상으로하고, 성인이 핀 6-8의 재생에 효과를 볼되었습니다, 그러나이 방법은 전기 충격이 필요하고 많은 내부 장기로 인해 자신의 위치 나 때문에 전기에 대한 민감성에 하나 electroporate하기 어려운 중단. 직접 분사가 구조적 무결성을 방해 할 수 있기 때문에 또는 크기가 제한되어 있기 때문에 또한, 유충의 일부 조직에 직접 주사하기가 어렵습니다. finfold에 직접 분사 할 수 없기 때문에 유충의 꼬리 지느러미는 하나의 구조입니다. 따라서, 전기 천공 및 직접 분사에 대한 대안은 어느 주입 너무 작아서 또는 일렉트로 수없는 조직에서의 유전자를 표적으로 필요했다. 유생 꼬리 지느러미 재생시 특정 유전자의 기능을 대상으로 억제하기 위해, 우리는 모르 폴리 있도록 기존 기술을 수정 한늦은 무대 애벌레 꼬리 지느러미 재생하는 동안 유전자 기능의 ssessment. 이 방법은 함께 엔도 - 포터의 형질 전환 시약 (10)과 형광 태그가 morpholinos와의 심실 전달 9를 사용한다. 일단 심실에, 모르 폴리 노 - 엔도 - 포터 혼합물을 신속하게 혈관을 통해 유충에 걸쳐 퍼져 대상으로 이전에 불가능했을 조직을 입력합니다. 이 주입법은 특정 조직에서의 유전자를 표적으로하도록 변경 될 수 있으며, 아마도 현재의 유전자의 기능을 억제하는 역 유전 방법 부족한 다른 동물 모델에 적용 할 수있다. 따라서, 즉시 유충 단계에서 일반적으로 기관의 항상성 및 재생하는 동안 유전자의 기능을 연구하는 넓은 범위의 사용 가능성이있는 빠르고 쉬운 방법을 제공합니다.

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			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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			<td height="42" style="height:42px;width:216px;">Morpholino with 3&#180;fluorescein tag</td>
			<td style="width:171px;">Gene Tools Inc.&#160;</td>
			<td style="width:119px;">Customized</td>
			<td style="width:167px;">More information on www.genetools.com</td>
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			<td height="42" style="height:42px;width:216px;">Endo-Porter</td>
			<td style="width:171px;">Gene Tools Inc.&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">More information on www.genetools.com</td>
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			<td height="63" style="height:63px;width:216px;">Agarose</td>
			<td style="width:171px;">Serva</td>
			<td style="width:119px;">120623</td>
			<td style="width:167px;">For pouring plates to place the fish during injections and imaging</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tricane (L-Ethyl-m-amino-benzate-methane sulfonate)</td>
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			<td style="width:167px;">For anesthesia</td>
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			<td height="63" style="height:63px;width:216px;">E3 medium (5 mM NaCl; 0.17 mM KCl; 0.33 mM CaCl2; 0.33 mM MgSo4)</td>
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			<td style="width:119px;"></td>
			<td style="width:167px;">For larval husbandry</td>
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			<td style="width:171px;">Sarstedt</td>
			<td style="width:119px;">821.472</td>
			<td style="width:167px;">For placing the fish during injections and imaging</td>
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			<td height="21" style="height:21px;width:216px;">&#160;Equipment</td>
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			<td height="42" style="height:42px;width:216px;">Thin Wall Glass Capillaries</td>
			<td style="width:171px;">World Precision Instruments, Inc.&#160;</td>
			<td style="width:119px;">TW100F-3</td>
			<td style="width:167px;">For preparing injection needles</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microloader pipette tips</td>
			<td style="width:171px;">Eppendorf</td>
			<td style="width:119px;">5,242,956,003</td>
			<td style="width:167px;">For loading the needle</td>
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			<td height="21" style="height:21px;width:216px;">Pasteur Pipettes (3 ml/1 ml)</td>
			<td style="width:171px;">Brand</td>
			<td style="width:119px;">747760/ 74775</td>
			<td style="width:167px;">For transfering larva</td>
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			<td height="42" style="height:42px;width:216px;">Flaming / Brown&#160; needle puller&#160;</td>
			<td style="width:171px;">Sutter</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">More information on www.sutter.com</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Watchmaker (Dumont) Tweezers (size 5)</td>
			<td style="width:171px;">World Precision Instruments, Inc.&#160;</td>
			<td style="width:119px;">501985</td>
			<td style="width:167px;">To brake the needle before sharpening</td>
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			<td height="42" style="height:42px;width:216px;">Dissecting microscope</td>
			<td style="width:171px;">Olympus, Leica, Zeiss</td>
			<td style="width:119px;">Varies with the manufacturer</td>
			<td style="width:167px;">Part of the whole injection setup</td>
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			<td height="63" style="height:63px;width:216px;">Fluorescence camera</td>
			<td style="width:171px;">Olympus, Leica, Zeiss</td>
			<td style="width:119px;">Varies with the manufacturer</td>
			<td style="width:167px;">To image the fluorescence after injeciton</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PicoNozzle kit (microinjector holder)</td>
			<td style="width:171px;">World Precision Instruments</td>
			<td style="width:119px;">5430-12</td>
			<td style="width:167px;">For microinjections</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pneumatic PicoPump (microinjector)</td>
			<td style="width:171px;">World Precision Instruments</td>
			<td style="width:119px;">SYS-PV820</td>
			<td style="width:167px;">For microinjections</td>
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reverse genetic morpholino approach using cardiac ventricular injection to transfect multiple difficult-to-target tissues in the zebrafish larva

제브라 피쉬는 세포와 기관 및 부속 기관 재생의 분자 생물학을 이해하는 중요한 모델입니다. 그러나, 역 유전학을 사용하는 분자 전략은 아직 충분히 제브라 피쉬 배아 후 유생 단계 동안 재생 또는 조직의 항상성의 유전자 기능을 평가하기 위해 개발되지 않은, 그리고 제브라 피쉬의 유충 내에서 여러 조직을 대상으로하기 어렵다. 유전자 특정 morpholinos와의 뇌 실내 주사는 유 전적으로 이러한 단계에서 일시적으로 통제 된 방식으로 zebrafish의 유전자를 표적으로하는 현재의 무능력에 대한 대체 방법을 제공합니다. 이 방법은 이전과 같은 유생의 꼬리 지느러미, 종종 비 침습적 조직 재생을 연구하는 데 사용되는 구조에서와 도달하기 불가능했던 구조 등 신체 전반에 걸쳐 다양한 조직에 완전 분산 모르 폴리 노의 연속 편입 할 수 있습니다. 애벌레 finfold 재생하는 동안 활성화 몇 가지 유전자는 프리젠됩니다성인 척추 조직의 재생에 t, 그래서 애벌레는 성인의 재생을 이해하는 데 유용한 모델입니다. 이 모르 폴리 노 분산 방법은 애벌레 조직의 재생뿐만 아니라 배아 후 조직의 항상성을 조절하는 다른 생리 현상에 필요한 유전자를 빠르고 쉽게 식별 할 수 있습니다. 따라서, 이러한 전달 방법은 배아 발생 후의 유전자 기능의 평가에 시간적 제어에 대한 현재 필요한 전략을 제공한다.

(그림 3A) 등쪽으로 접근하면 날카로운, 경 주사 바늘을 ​​쉽게 제브라 피쉬의 유충의 심장 심실에 배치됩니다. 마음은 바늘 (그림 3B 및 3C)의 존재에도 불구하고 펌핑을 계속하고 혈액의 흐름이 유지됩니다. 조심 주사는 심장 (그림 3E)에 모르 폴리 노의 분사에도 불구하고 심실 또는 심장 수축 (그림 3D)의 형태를 방해하지 않습니다....

Watch this Scientific Journal Video about Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva at JoVE.com

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

이러한 유전자의 특정 morpholinos와의 뇌 실내 주사를 사용하여 조직 재생 등의 개발 및 생리 학적 항상성 이후 단계에서 유전자 기능을 평가하는 제브라 피쉬에 대한 적응력이 역 유전 방법.

Zebrafish의 유충의 여러 어려운 대상 조직을 형질 심장 심실 사출을 사용하여 유전 모르 폴리 노의 접근을 반전

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to ...

reverse-genetic-morpholino-approach-using-cardiac-ventricular

Research

JoVE Journal

Biology

10.5K 조회수.  Technische Universität Dresden.  이러한 유전자의 특정 morpholinos와의 뇌 실내 주사를 사용하여 조직 재생 등의 개발 및 생리 학적 항상성 이후 단계에서 유전자 기능을 평가하는 제브라 피쉬에 대한 적응력이 역 유전 방법. 

비디오: Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

Zebrafish 태아의 mRNA와 Morpholino의 안티 센스 Oligonucleotides의 Microinjection.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

연구

생물학

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Zebrafish의 두뇌 뇌실 주입

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

성인 Zebrafish에서 레트로 - 궤도 분사

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

고효율 기본 셀을 Transfect하는 진 Pulser Electroporation MXcell의 시스템을 사용하여

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

유전자 Inducible의 운명 매핑에 대한 실용적인 접근법 : 셀을 선택하고 추적하는 비주얼 가이드 VIVO에서</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

Embryogenesis 동안 중복 기능을 테스트하기 위해 역방향 유전자 접근

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

성인 Zebrafish에 Intraperitoneal 사출

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

성인 Zebrafish의를 Gavaging

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Zebrafish의에서 콩팥 세포 독소 미세 주입은 급성 신장 손상을 모델링하기

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible cytoplasmic compartments. Another hurdle is the tight apposition of myofibers to the surrounding tissues. Because the process of tissue fixation and paraffin embedding leads to the shrinkage of muscle fibers, freezing is an optimal means of hardening muscle tissue for sectioning. However, a commonly encountered issue, the formation of ice crystals, occurs during the preparation of frozen sections because of the high water content of muscle. The protocol presented here first describes a simple and efficient method for properly freezing muscle tissues by immersing them in liquid nitrogen. The problem with using liquid nitrogen alone is that it causes the formation of a nitrogen gas barrier next to the tissue, which acts as an insulator and inhibits the cooling of the tissues. To avoid this "vapor blanket" effect, a new cryovial was designed to increase the speed of liquid flow around the tissue surface. This was achieved by punching a total of 14 inlet holes in the wall of the vial. According to bubble dynamics, a higher rate of liquid flow results in smaller bubbles and fewer chances to form a gas barrier. When liquid nitrogen flows into the cryovial through the inlet holes, the flow velocity around the tissue is fast enough to eliminate the gas barrier. Compared to the method of freezing muscle tissues using pre-chilled isopentane, this protocol is simpler and more efficient and can be used to freeze muscle in a throughput manner. Furthermore, this method is optimal for institutions that do not have access to isopentane, which is extremely flammable at room temperature.

This protocol describes a procedure to freeze muscle tissues by plunging them directly into liquid nitrogen. This protocol also highlights a new cryovial that can avoid the &#34;blanket effect&#34; of nitrogen gas when liquid nitrogen contacts the tissue surface of a specimen.

근육 조직이 직접 액체 질소에 몰입 할 때 이슈 결빙 멀티 홀 Cryovial 제거

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible ...