우리는 pDB600 플라스미드의 종류 선물 우수한 수산 축산에 대한 양 폴라 로이, 그리고 스티브 Ekker 감사드립니다. 이러한 연구 NHGRI에서 SF에 부여로 투자했다.

1. 선택하고 테스트하기위한 보존 시퀀스의 클로닝 <ol><li> 하나는 첫번째 기능 테스트를 위해 잠재적인 규제 시퀀스를 식별해야합니다. 우리는 순서를 선택하는 기준으로 진화 순서 보존에 의존하고, 가장 많이 UCSC 게놈 브라우저 (http://genome.ucsc.edu/cgi-bin/hgGateway)에 트랙으로 액세스할 수있는 알고리즘 PhastCons를 사용합니다. 그러나, 수종에 걸쳐 순서 절약을 평가하기 위해 사용할 수있는 여러 가지 다른 알고리즘이 있습니다. </li><li> 일단 우리가 우리의 시퀀스를 선택, 우리는 게놈 DNA에서 각 순서를 증폭하기 위해 primers를 디자인. Primers는 양쪽에있는 보존 지역 플러스 30 개 이상의 기본 쌍을 증폭하기 위해 선택되어야합니다. </li><li> 선택된 시퀀스의 증폭을 위해 우리는 다카라 LA TaqTM 시스템을 사용합니다. 기타 polymerases가 작동하지만, 그것은 증폭 동안 소개 오류의 가능성을 최소화하기 위해, 교정 기능이있는 효소를 포함하는 혼합을 사​​용하는 것이 중요합니다. 우리는 아가로 오스 겔 전기 영동에 의해 amplicon의 크기를 확인합니다. </li><li> 우리는 PCR 8/GW/TOPO TA 복제 키트 (Invitrogen)가 게이트웨이 항목 클론을 생성, attL 재조합 사이트를 포함하는 입력 벡터로 PCR 제품을 복제하기 위해 사용합니다. 복제 반응은 신선한 PCR 제품과 함께보다 효율적이기 때문에 PCR의 하루 이내에 복제를 수행하는 것이 최선입니다. DH5α의 변종의 변환은 spectinomycin 저항 선택에 의해 다음이 수행됩니다. </li><li> 우리는 삽입을 공개 EcoRI과 플라스미드의 ~ 500ng의 소화에 의해 TA 복제의 제품을 확인하고, 아가로 오스 겔 전기 영동에 의해 삽입의 크기를 확인합니다. 우리는 삽입 순서와 방향을 확인하는 순서 권장, 증폭에 사용된 primers는 시퀀싱을 위해 사용할 수 있습니다. </li><li> 항목 복제에서 비 코딩 보존 시퀀스는 게이트웨이 LR Clonase II 효소 믹스 (Invitrogen)를 사용하여 우리의 게이트웨이 준비 대상 벡터, pGW_cfosGFP1에 LR의 재조합에 의해 shuttled 있습니다. DH5α의 변종의 변환은 암피실린 저항의 선택에 의해 다음이 수행됩니다. </li><li> 우리는 삽입을 공개 EcoRV와 플라스미드의 ~ 500ng의 소화에 의해 LR 재조합의 제품을 확인하고, 아가로 오스 겔 전기 영동에 의해 삽입의 크기를 확인합니다. 정확한 복제가 확인되면, 우리는 Qiagen HiSpeed​​를 사용하여 플라스미드 DNA 플라스미드 ® 미디 키트를 준비합니다. 우리는 더 이상 제조 업체의 프로토콜에 의하면, QIAquick PCR의 정제 키트를 사용하여 플라스미드를 정화, 30 μL RNase 무료 물로 DNA를 elute. 플라스미드는 μL / NG와 4에 저장된는 ° C 이전에 주사 125의 농도로 희석합니다. </li><li> RNA 인코딩 기능 Tol2 transposase 효소는 pDB600 plasmid2에서 체외에서 베꼈는데 있습니다. 우리는 Qiagen 미디 준비 키트를 사용하여 플라스미드를 정화하고 XbaI와 10-20 μg을 linearize. mRNA 합성이 mMESSAGE mMACHINE 키트 프로토콜 (앰비온) 다음과 같은 수행됩니다. </li><li> 우리는 정화 및 키트 지침에 따라 RNA를 침전. 우리는 RNase 무료로 물 속에, 마지막 ~ 1μg/μl의 농도, 또는 하나의 반응 20 μl로 RNA를 resuspend하고, UV 분광 광도법으로 정량. 우리는 또한 전체 길이 전송을 확인하는 아가로 오스 겔 전기 영동으로 RNA의 ~ 1μg을 분석. 표준 태 또는 TBE 젤이 분석에 적합하지만, 전사 키트에 포함되어있는 denaturing 샘플 버퍼 키트의 지시에 따라 사용해야합니다. </li><li> 우리는 알려진 플라스미드 (그림 1)과 주사를 수행하여 효험에 대한 transposase RNA의 각각의 새로운 배치를 테스트합니다. 확인 후, RNA는 작은 aliquots로 나뉘어 있으며, -80 ° C에서 저장, 반복 해동 및 개인 aliquots의 refreezing을 피할 수 있습니다. <img src="/files/ftp_upload/1722/1722fig1.jpg" alt="그림 1" /> 그림 1. Zebrafish의 배아는 제어할 수 있습니다. (위) 브라이트 이미지 (아래) GFP 형광 이미지로 마우스 Sox10 확장기로 주입. transposase RNA의 각 새로운 배치는 효능에 대해 테스트됩니다. </li></ol> 2. 모자이크 유전자 변형 분석을 위해 zebrafish 배아의 Microinjection <ol><li> 우리는 그대로 chorions을 뚫을 상당히 날카로운 테이퍼와 강력한 팁을 얻을 수 있도록 설계된 프로그램, 셔터 P - 97 Micropipette의 풀러에서 1.2 mm OD 필라멘트 모세관 유리 microinjection을 위해 바늘을 가져옵니다. 우리는 깨끗한 면도날와 마이크로 미터의 슬라이드를 사용하여 약 15 μm의의 외경에 stereomicroscope 아래 손으로 팁을 휴식. 바늘은 주사 전날했고, 깨끗하게 유지하는 대상 지주 접시에 저장할 수 있습니다. </li><li> 우리는 표준 conditions3 아래 빛을 14 시간과 함께, 일반 조명 어두운주기에 우리의 zebrafish를 유지합니다. 일 이전에 수행 microinjections에, 우리는 작은 사육 탱크, 기본 탱크로 구성된 각 홈 붙이 삽입 및 플라스틱 뚜껑에 초과 matings을위한 물고기를 설정합니다. 우리는 세 여성 또는 t 당 두 남자를 배치병렬 행을 ank. </li><li> microinjections 아침에 빛을주기가 시작 직후, 우리는 계란 생산의 클린 시스템 처리 물에서 남성과 여성을 결합 시작합니다. 자주 생선을 확인하고, 부설의 몇 분 이내에 달걀을 수집, 잘 동기 배치를 얻을 것이 중요합니다. 계란 생산은 아침 내내 신선한 물고기의 그룹을 결합하기 위해 계속하여 2 ~ 세 시간 동안 유지하실 수 있습니다. </li><li> 넓은 구멍으로 5 1 / 4 &quot;유리 파스퇴르 피펫은 라텍스 전구가 장착되어, 우리가 50 배아의 그룹 부분 엠브료 Medium2 가득 60 X 15mm 페트리 요리,에 수집된 배아를 정렬하고, 마르크 시간에 수집 각 접시의 뚜껑. </li><li> 물고기가 누워 시작되면, 우리는 얼음에 microfuge 튜브에 다음을 혼합하여 신선한 사출 솔루션을 준비 : <table border="1"><tbody><tr><td> 구성 요소 </td><td></td></tr><tr><td> Transposon 플라스미드 주식 (125ng/μl) </td><td> 1μl </td></tr><tr><td> Transposase RNA 주식 (175ng/μl) </td><td> 1μl </td></tr><tr><td> 페놀 레드 주식 (H 2 O 2 %) </td><td> 0.5μl </td></tr><tr><td> RNase 무료로 물 </td><td> 2.5μl </td></tr></tbody></table> 주사 바늘은 요리를 들고서도 잠자리 각 바늘의 광범위한 엔드에 주입 용액 500 NL의 방울을 pipetting으로 채워지게된다. 액체가 모세관을 통해 끝부분에 그림이 그려진 후에, 추가적인 솔루션은 1.5-2 μl 총 추가할 수 있습니다. 우리는 각 주사 솔루션을위한 최소한 두 바늘을 준비합니다. </li><li> 가득 바늘이 손에 구성하고 제조 업체의 지침에 따라 N2 탱크에 연결된 공압 피코 - 펌프 또는 유사 압력 주입기의 바늘 홀더 개최에로드됩니다. 우리는 마이크로 미터의 슬라이드에 광유로 퇴학 처분 방울의 직경을 측정하여 사출 볼륨을 보정합니다. 일반적으로 20 PSI의 압력으로 120 MS의 주입 시간은 약 1 NL의 비말을 낼 수 있지만, 바늘 직경 약간의 차이는 이러한 매개 변수에 영향을 미칠 것이며, recalibration가 바늘 사이에 필요할 수 있습니다. </li><li> 주사는 6 - 10X 배율에서 stereomicroscope에 따라 수행됩니다. 우리는 장소에 배아를 보유 chorion 통해 한 셀 또는 셀이 초기 단계에서 배아의 노른자에 지속적인 압력으로 주입 바늘을 밀어 벌금 포셉 한 쌍의을 사용합니다. 이상적으로, 우리는 그냥 blastomeres 아래의 노른자에서 바늘 끝의 위치. 사출 솔루션의 약 1 NL 퇴학과 바늘이 철회됩니다. 각각은 우리가 ≥ 200 배아를 주입 구축하십시오. 그것은 uninjected 제어 배아의 요리를 저장, 수집 태아의 각 클러치에 대해, 또한 매우 중요합니다. </li><li> 주사가 완료되면 우리는 unfertilized 계란, 손상된 배아, 그리고 실패 주사 (blastomeres에 없음 페놀 레드 배아) 제거하여 배아를 정렬하십시오. 그러면 ° C 적절한 시간까지 관찰을위한 28.5에서 배아 매체에서 배아를 품어. </li></ol> 3. 모자이크 표현 패턴 분석 <ol><li> 적절한 배양 시간에 따라, 우리는 형광에 대한 주입 배아를 검사하십시오. 타이밍은 내생 zebrafish의 유전자의 정상적인 표현 패턴에 따라 다르지만, 우리는 보통 처음 5 일간 게시물 수정을 통해 매일 봐요. </li><li> 우리는 epifluorescence에 대한 장착 올림푸스 MVX10의 macroscope에서 형광을위한 배아를 검사하지만, 낮은 전력 이미징 가능한 모든 유사한 현미경 작동합니다. 사망 또는 첫날에 비정상적으로 개발한 배아들 중 다수가있다면, 그들이 정상으로 보이는지되는 클러치에 대한 uninjected 컨트롤보세요. 컨트롤 정상 보면, 가장 일반적인 문제는 주입된 DNA의 순도 부족한 것입니다. 또한, 초기 단계의 배아는 주입 플라스미드의 총 금액을 구분하므로 그것은 부량가 부정확했고 너무 많은 DNA가 주입되었다 수도 있습니다. </li><li> 배아를 검사하면, 그들은 모두 주입 구조에 대한 모자이크 될 것을 명심하십시오. 그것은 표현의 예상 도메인이 작고 특히 경우, 태아의 전혀주의 깊게 볼 필요합니다. 또한 표현이 동적 될 수 있다고 기억, 1 일에 대한 강한 형광 하루에 2와 표현에 대해 처음 3 일 4 일 뭔가를 보여주고 있습니다 부정적인 태아로 사라진다 수 있습니다. </li><li> 우리는보다 광범위한 표현을 몇 가지 배아를 선택하고, photomicroscopy에 의해 형광의 패턴을 문서에 다음을 사용합니다. 관찰 5 일 후 배아가 시설에 반환되어야하며 주식으로 키웠습니다. 몇 개월에서, 성인은 transgenes의 germline 전송에 대한 검사를하실 수 있습니다. </li></ol> 4. 검색 결과 우리는 넓은 V를 참조하십시오분석중인 향상제 요소에 따라 패턴과 표현의 수준 ariety. 우리는 일반적으로 매우 구체적인 조직 표현 패턴에 관심이 있으며, 종종 골격 표현 유전자에 초점을 맞추고 있습니다. 그리고 종종 골격 표현 유전자에 초점을 맞추고 있습니다. 그림 2는 연골과 뼈의 표현을 규제 강화와 함께 우리가 주입된 배아에서 관찰이 모자이크 표현 패턴의 예제를 보여줍니다. 마지막으로, 테스트 시퀀스의 상당한 부분은 조직 구체적인 표현을 규제하지 않습니다. 이러한 경우, 우리는 대부분 거의 형광, 배아 당 아마도 몇 흩어져있는 세포를 참조하십시오. 적게, 우리는 오직 이소성 표현, 표피 및 골격 근육 (그림 3)의 두 가지 일반적인 사이트에, 형광 볼 수 있습니다. <img src="/files/ftp_upload/1722/1722fig2.jpg" alt="그림 2" /> 그림 2. 주입 배아에서 관찰 연골과 뼈 표현 패턴의 예. <img src="/files/ftp_upload/1722/1722fig3.jpg" alt="그림 3" /> 그림 3. 유전자와 표현의 규정에 향상제의 상대의 위치 사이에 약간의 상관 관계가있다. 테스트 시퀀스의 상당한 부분은 조직 구체적인 표현을 규제하지 않습니다. 이들은 종종 거의 형광을 가지고, 또는 이소성 표현 두 가지 일반적인 사이트에있을 수 있습니다 : 표피와 골격 근육. (위) 브라이트 이미지 (아래) GFP 형광 이미지.

<ol>
	<li>Fisher, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+biological+relevance+of+putative+enhancers+using+Tol2+transposon-mediated+transgenesis+in+zebrafish.">Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish.</a> Nat Protoc. 1 (3), 1297-12 (2006).</li><li>Balciunas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+a+high+cargo-capacity+transposon+for+genetic+applications+in+vertebrates.">Harnessing a high cargo-capacity transposon for genetic applications in vertebrates.</a> PLoS Genet. 2 (11), e169-169 (2006).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book. , (1995).</li></ol>

우리는 zebrafish transgenesis를 사용하여 잠재적인 그때의 효율적인 분석을 위해 저희 실험실에서 사용되는 접근 방식을 증명하고있다. 대부분의 자주, 우리는 인간의 유전자와 연관된 조직 고유의 강화를 찾는이 방법을 사용합니다. 명백한 순서 상동의 부족에도 불구하고 인간과 물고기가 아닌 코딩 순서를, 우리가이 접근 방식이 우리에게 관심의 유전자에 대한 강화를 식별 보통 성공 것을 발견했?...

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<td>Takara LA Taq</td>
<td>&nbsp;</td>
<td>Takara Bio Inc</td>
<td>RR02M</td>
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<td>pCR8/GW/TOPO TA Cloning Kit</td>
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<td>Invitrogen</td>
<td>K2500-20</td>
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<td>Gateway LR Clonase II enzyme Mix</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11791-100</td>
<td>&nbsp;</td>
<tr>
<td>Library efficiency competent cells DH5&alpha;</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12535-019</td>
<td>&nbsp;</td>
<tr>
<td>Library efficiency competent cells DB3.1</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11782-018</td>
<td>&nbsp;</td>
<tr>
<td>mMESSAGE mMACHINE Kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM1340</td>
<td>&nbsp;</td>
<tr>
<td>HiSpeed Plasmid Midi Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12643</td>
<td>&nbsp;</td>
<tr>
<td>QIAquick PCR Purification Kit </td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28104</td>
<td>&nbsp;</td>
<tr>
<td>Flaming/Brown Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Pneumatic PicoPump</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>SYS-PV820</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

mosaic zebrafish transgenesis for evaluating enhancer sequences

많은 다른 종류의와 함께 인간 게놈 순서의 완성, 비 코딩 시퀀스에 특정 기능을 ascribing의 도전을 강조했다. 게놈의 비 코딩 분율에 의해 실시 하나의 두드러진 기능은 유전자 전사를 조절하는 것입니다,하지만, 널리 기본 DNA 시퀀스에서 CIS - 규제 요소를 예측하는 더 효과적인 방법은 없습니다. 우리는 기능 zebrafish transgenesis을 통해 잠재적인 CIS - 규제 요소를 평가하는 효율적인 프로토콜을 개발했습니다. 그것은 공간과 시간적 유전자 조절에 대한 정보를 제공 이후 우리의 접근 방식은, 발달 중요한 유전자에 대한 세포 문화 기반 기술에 비해 상당한 장점을 제공합니다. 반대로, 그것은 빠르고 저렴 유전자 변형 생쥐와 비슷한 실험보다, 우리는 정기적으로 인간 게놈으로부터 격리 시퀀스 프로그램을 적용해야합니다. 여기 복제 시퀀스에 대한 시퀀스 보호 및 프로토콜에 기초하고 zebrafish 배아에 그들을 microinjecting 테스트를위한 요소를 선택하기 위해 방법을 보여줍니다.

Watch this Scientific Journal Video about Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences at JoVE.com

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

우리는 발달 규제 유전자의 잠재적인 확장기 요소를 찾아서 모자이크 zebrafish의 transgenesis 통해 기능을 평가하기 위해 방법을 보여줍니다.

향상제 시퀀스를 평가하기위한 모자이크 Zebrafish의 Transgenesis

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non ...

mosaic-zebrafish-transgenesis-for-evaluating-enhancer-sequences

Research

JoVE Journal

Biology

13.9K 조회수.  University of Pennsylvania. 우리는 발달 규제 유전자의 잠재적인 확장기 요소를 찾아서 모자이크 zebrafish의 transgenesis 통해 기능을 평가하기 위해 방법을 보여줍니다.

비디오: Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

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 ...

연구

생물학

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

RNAi는 Necromenic 선충류의 Microinjection에 의해 유전자 최저 및 Transgenesis을 중재 Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Morpholinos의 생체내의 Electroporation에서 성인 Zebrafish의 망막에

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

생성중 성인 Zebrafish 테일 핀으로 Morpholinos의 생체내 Electroporation에

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Zebrafish 배아 골격근의 레이저 쏜 부상

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

모자이크 성인의 라이브 이미징 토마토 / GFP-FLP / FRT 방법<em&gt; 초파리</em&gt; 광 수용체 (photoreceptor)

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 108 cells/spermatogenic cell type from a starting population of 7-8 x 108 cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

STA-PUT 속도의 침전을 사용하여 정자 형성 세포 유형의 분리

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

동적 칼슘의 자동 분석<sup&gt; 2 +</sup이미지 시퀀스에서&gt; 신호

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity greatly impacts cell behavior to regulate normal and pathologic processes including cell proliferation, differentiation, adhesion signaling and directional migration. In this regard, the innate ability of certain cell types to migrate towards a stiffer, or less compliant matrix substrate is referred to as durotaxis. This phenomenon plays an important role during embryonic development, wound repair and cancer cell invasion. Here, we describe a straightforward assay to study durotaxis, in vitro, using polydimethylsiloxane (PDMS) substrates. Preparation of the described durotaxis chambers creates a rigidity interface between the relatively soft PDMS gel and a rigid glass coverslip. In the example provided, we have used these durotaxis chambers to demonstrate a role for the cdc42/Rac1 GTPase activating protein, cdGAP, in mechanosensing and durotaxis regulation in human U2OS osteosarcoma cells. This assay is readily adaptable to other cell types and/or knockdown of other proteins of interests to explore their respective roles in mechanosignaling and durotaxis.

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

평가 셀 Mechanosensing 및 Durotaxis에 대한 단순화 된 시스템<em&gt; 체외</em

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...

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 ...