저자는 실험실에서이 기술을 개발 로라 앤 로우 어리 감사하고 싶습니다. HS는 NIHMH70926에 의해 지원됩니다. 제니퍼 구츠맨은 CSBi / 머크 박사 친교에 의해 지원되었다.

프로토콜 1 부 : microinjection에 대비하여 <ol><li> 서터 인스 트루먼 트의 바늘 풀러를 사용하여 모세 바늘을 떼어하여 사출을위한 준비. </li><li> 형광 염료 (예 : 텍사스 - 레드 Dextran 등)로 바늘을 입력합니다. </li><li> micromanipulator과 microinjection 장치에 바늘을 탑재합니다. </li><li> beveled 팁을 만들 수있는 각도로 적절한 크기로 바늘을 잘라요. </li><li> 드롭 크기를 측정하고 기름 주입 당 1 2nl 사이에 있는지 확인합니다. </li></ol> 2 부 : 배아 준비 <ol><li> 관심의 무대 30 분만 일찍 배아를 주사. 예를 들어, 24 시간 게시물 수정 (HPF)에서 심실을 공부하기 위해, 23.5 HPF에서 배아를 주입. 킴멀 외에 따라 배아를 무대. (4). </li><li> stereomicroscope에서 신중하게 집게를 사용하여 태아의 chorion를 제거합니다. </li><li> 1% 아가로 오스와 코팅 플라스틱 접시를 사용하여 플라스틱 1-200 μl 피펫 팁있는 아가로 오스의 구멍을 찌르지. 조심스럽게 집게를 사용하여 구멍의 아가로 오스의 플러그를 제거합니다. </li><li> 구멍 아가로 오스 요리에 배아 미디어의 배아를 전송합니다. </li><li> 미디어에 추가 tricaine은 (웨스 터 필드 (5)에 따라 만들어진)은 배아를 마취 및 실험 도중 움직임을 방지하기 위해. </li><li> 부드럽게 구멍에 배아 꼬리를 장소, 사출 장치는 배아의 후부 측면에 있도록 동양 그것. </li></ol> 파트 3 : 뇌의 뇌실을 주입 <ol><li> 뇌의 뇌실을 삽입하려면 바늘 끝이 높은 전원 배아로보기 같은 분야에서되도록 미세 조작 설정을 주선해드립니다. </li><li> 조심스럽게 아래의 뇌 조직을 누르지 말고 r0/r1 hingepoint 단 후부 hindbrain의 얇은 roofplate를 통해 바늘을 넣어. </li><li> 주입은 충분히 완전히 심실을 채우기 위해 염료. 그것은 배아의 단계에 따라 뇌실 공간을 채우기 위해 여러 가지 주사 걸릴 수 있습니다. </li></ol> 4 부 : 영상 <ol><li> 깨끗한 1퍼센트 아가로 오스 - 코팅 접시를 사용하여 요리에 새로운 구멍을 찌르지, 플러그를 제거하고 배아를 마취하고 움직임을 방지하기 위해 tricaine를 추가합니다. </li><li> 지느러미 이미지의 구멍과 위치에 뇌실 - 주입된 배아의 꼬리를 놓습니다. </li><li> 전송 빛을 사용하여 이미지를 가져가라. </li><li> 그것은 배아 또는 현미경을 이동할하는 것은 매우 중요합니다. </li><li> 현미경의 설정을 변경하고 형광등과 이미지를 가져가라. </li><li> 전송 빛 형광 불빛 모두 측면 이미지를 취할 배아를 재조 정할. </li><li> 포토샵에서 이미지 처리를 위해 TIFF 파일로 이미지를 저장합니다. </li></ol> 제 5 부 : 이미지 처리 <ol><li> 포토샵에서 이미지를 처리​​하기 위해, 전송 조명과 같은 위치에서 찍은 같은 배아의 형광 이미지를 엽니다. 모든 설정은 각 이미지의 동일해야합니다. </li><li> 전송 빛의 이미지에 귀하의 형광 이미지를 드래그하고이를 정확하게 라인. </li><li> 형광 이미지가 선택된 상태에서 &quot;이미지&quot;, &quot;조정 선택&quot;다음 &quot;색상 바꾸기&quot;과 흰색에 검정색 배경을 변경할 수 있습니다. </li><li> 형광 이미지 색상의 균일 보인다 때까지 &quot;바꾸기 컬러&quot;창에서 오른쪽에있는 &quot;fuzziness&quot;요소를 조정합니다. </li><li> &#39;레이어&#39;창에서 오버레이 이미지를 볼 수 있습니다 &quot;곱하기&quot;을 선택합니다. 당신의 뇌실 공간 이미지가 정확하게 전송 빛의 이미지와 일치해야합니다. </li><li> 다른 이미지에 대해이 파일과 반복을 저장합니다. </li></ol> 대표 결과 / 결과 잘못된 사출 이미지의 예제 EH 표시하는 동안 적절한 뇌실 주입 이미지는 1 패널 AD 그림에 표시됩니다. 적절한 주사는 날카로운 가장자리와 비 확산 염료 있습니다. 바늘이 꽂혔어 너무 멀리 삽입 아래의 뇌 조직을 치는 경우에 발생합니다 잘못된 주사, 뇌실 공간의 볼 수 밖에 염료와 노른자가 발생할 수 있습니다. <img src="/files/ftp_upload/1218/1218figure1.png" alt="그림 1" width="600" height="742" /> 그림 1. 25 HPF 야생 유형 태아의 뇌실 주사. (AD) 올바른 주사 방법과 (EH) 잘못된 주입 방법은 예제로 표시됩니다. 해당 형광 이미지 (A, E) 도설 브라이트 이미지 (B, F)와 같이 정확하고 잘못된 모두 주입 방법에 대한 형광 및 브라이트 이미지의 오버레이 (C, G). 화살표는 염색이 잘못된 뇌실 주입 ( &quot;문제 해결&quot;을 참조)로 인해 뇌의 뇌실 공간 넘어 간 영역을 나타냅니다. 앞의 모든 이미지 왼쪽입니다. 문제 해결 : <table border="1"><tbody><tr style="font-weight:bold; font-size:16px;"><td> 문제 </tD&gt; <td> 원인 </td><td> 치료제 </td></tr><tr><td> 당신은 바늘로 배아를 부수 있습니다. </td><td> 당신의 바늘이 너무 무딘거나 끝이 너무 큽니다. </td><td> 새 바늘로 시작하여 그것을 멀리 다시 휴식하지 않습니다. 베벨은 바늘 주사기 바늘처럼 날카로운 만들 수 있습니다. </td></tr><tr><td> 염료 (그림 1E - H 참조) 배아와 노른자에 걸쳐있다. </td><td> 당신의 바늘은 조직을 통해 가서 뇌실 공간에서 체류하지 않았다. </td><td> 당신의 바늘을 충분히 예리하지 않을 수 있습니다 당신이 주사의 속도를 제어 수 없습니다 그래서. </td></tr><tr><td> 염료는 forebrain 너무 희미하거나하지 않습니다. </td><td> 당신은 주사만큼 충분히 높은 압력을 사용하지 않았거나 바늘은 멀리 당신이 염료를 주입 때 forebrain에 도달하는 앞쪽에되지 않았습니다. </td><td> 귀하의 분사 압력을 높이거나 사출 동안 바늘이 약간 더 앞쪽에 놓습니다. 자세한 내용은 forebrain에 필요한 경우에는 midbrain - hindbrain 경계를 통해 바늘을 넣을 수 있습니다. 당신은 또한 단순히 영상 전에 더욱 확산하는 염료에 대해 조금만 기다려 수 있습니다. </td></tr><tr><td> 당신의 형광 이미지를 포토샵에서 색상 교체 후 가장자리 주위에 어두운 있습니다. </td><td> 당신은 색상 바꾸기 창에서 &quot;fuzziness&quot;설정을 변경하려면 잊어버렸습니다. </td><td> &quot;fuzziness&quot;그 색상 바꾸기 창에서 오른쪽에있는 모든 방법을 이동합니다. </td></tr><tr><td> 바늘이 찔린 hindbrain의 roofplate하지 않습니다. </td><td> 당신의 주사 바늘을 제대로 절단되지 않습니다. </td><td> 새로운 바늘을합니다. 당신이 주사를 많이하고있다면 그들이 무딘 얻을 수있는 몇 가지 바늘이 필요할 수 있습니다. </td></tr></tbody></table>

<ol>
	<li>Gutzman, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+zebrafish+midbrain-hindbrain+boundary+constriction+requires+laminin-dependent+basal+constriction.">Formation of the zebrafish midbrain-hindbrain boundary constriction requires laminin-dependent basal constriction.</a> Mech. Dev. 125, 974-983 (2008).</li><li>Lowery, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+Classification+of+Zebrafish+Brain+Morphology+Mutants.">Characterization and Classification of Zebrafish Brain Morphology Mutants.</a> Anat. Rec. (Hoboken). 292, 94-106 (2009).</li><li>Lowery, L. A., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+formation+of+zebrafish+brain+ventricles+occurs+independently+of+circulation+and+requires+the+nagie+oko+and+snakehead/atp1a1a.1+gene+products.">Initial formation of zebrafish brain ventricles occurs independently of circulation and requires the nagie oko and snakehead/atp1a1a.1 gene products.</a> Development. 132, 2057-2067 (2005).</li><li>Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203, 253-310 (1995).</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: A guide for the laboratory use of zebrafish. , (1995).</li></ol>

이 비디오에서 우리는 zebrafish 두뇌 ​​심실 개발에 형광 염료 (텍사스 - 레드 Dextran)를 주입하는 방법을 보여줍니다. 이 방법은 주변 neuroepithelium 달리 뇌 뇌실 공간을 시각화하는 데 사용과 뇌실 공간의 형태뿐 아니라 주변의 뇌 조직의 모양을 결정하는 매우 유용합니다. 우리가 더 나은 살아있는 배아에서 시간이 지남에 따라 뇌 뇌실 형성 및 뇌 morphogenesis의 과정을 이해할 수 있습니다. 이 기술?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Forceps</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11252-20</td>
<td>&nbsp;</td>
<tr>
<td>Capillary Tubes (needles)</td>
<td>Tools</td>
<td>Frederick Haer and Co.</td>
<td>30-30-1</td>
<td>&nbsp;</td>
<tr>
<td>Agarose-Coated dishes</td>
<td>Tools</td>
<td>Agarose (Seakem GTG; Lonza) Dishes (Corning) </td>
<td>Agarose (50027) Dishes (430166) </td>
<td>Make agarose approximately 2-3 mm deep in the dish depending on the stage of embryo you are injecting</td>
</tr>
<tr>
<td>Dissecting Microscope</td>
<td>Microscope</td>
<td>Zeiss Stereomicroscope Stemi SV 6 </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microscope Camera with Fluorescence capabilities and with image capture software</td>
<td>Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1-200 &mu; pipette tips</td>
<td>Tools</td>
<td>Tip One, US Scientific</td>
<td>1111-0806</td>
<td>These are the perfect size to poke the holes in the agarose and have the embryos fit perfectly.</td>
</tr>
<tr>
<td>Photoshop</td>
<td>Image Analysis</td>
<td>Adobe</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Embryo Loop</td>
<td>Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Make these yourself with a glass pipette and hair or fishing line and seal with beeswax.</td>
</tr>
<tr>
<td>Microinjector</td>
<td>Tools</td>
<td>Medical Systems Research Products Harvard Apparatus</td>
<td>PL1-100</td>
<td>&nbsp;</td>
<tr>
<td>Needle Puller</td>
<td>Tools</td>
<td>Sutter Instruments</td>
<td>&nbsp;</td>
<td>Settings (Heat 785, Pull 70, Velocity 40, Time 70).</td>
</tr>
<tr>
<td>Micromanipulator</td>
<td>Tools</td>
<td>Narishige</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent dye Texas-Red Dextran 70,000MW</td>
<td>Reagent</td>
<td>Molecular Probes</td>
<td>D1830</td>
<td>Other Molecular weights are available if desired.</td>
</tr>
<tr>
<td>Tricaine (3-aminobenzoic acid ethyl ester)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-5040</td>
<td>&nbsp;</td>
<tr>
<td>Embryo Media</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>According to Westerfield (5)</td>
</tr>		</tbody>
		</table>
		</div>

zebrafish brain ventricle injection

배아 두뇌 개발하는 동안 적절한 두뇌 뇌실 형성은 정상적인 뇌 기능이 필요합니다. 뇌 심실은 뇌척수로 가득하는 두뇌 내의 고도 보존 충치 있습니다. 그것이 두뇌 뇌실 형성의 결과로 액체로 채우고있는 동안 zebrafish에서 신경 튜브 형성 후, neuroepithelium는 constrictions과 주름 일련의를 겪습. 뇌실 형성 과정과 동시에 발생하는 neuroepithelial 모양의 변화를 이해하기 위해서, 우리는 뇌 조직에 비해 뇌실 공간을 시각화하는 방법이 필요했습니다. 그러나, 투명한 zebrafish의 배아의 본질은 어려운 뇌실 우주에서 조직을 차별화 수 있습니다. 따라서, 우리는 뇌실 공간이 형광 염료 가득한과 브라이트 및 형광 현미경으로 몇 군데있는 두뇌 뇌실 사출 기술을 개발했습니다. 브라이트과 형광 이미지는 다음 처리와 포토샵으로 겹쳐있다. 이 기법은 브라이트 이미지의 neuroepithelium의 형태 비교, 형광 염료와 뇌실 공간의 시각화를위한 수 있습니다. zebrafish의 뇌 뇌실 주입은 초기 애벌레 단계를 통해 18시간 게시물 수정에서 근무하실 수 있습니다. 우리는뿐만 아니라 두뇌 morphogenesis의 돌연변이 (1-3)를 특성화 같이 뇌실 뇌 형성과 morphogenesis 우리의 연구에 광범위하게이 기술을 사용했습니다.

Watch this Scientific Journal Video about Zebrafish Brain Ventricle Injection at JoVE.com

Zebrafish Brain Ventricle Injection

신경 튜브 형성 후, neuroepithelium은 수축과 튜브가 배아 두뇌 심실을 형성하기 위해 배아 뇌척수 (eCSF)로 채우고 동안 주름. 우리는 더 나은 라이브 배아에서 neuroepithelial 모양 대조적으로 액체 채워진 공간을 시각화이 뇌실 주입 기술을 개발했습니다.

Zebrafish의 두뇌 뇌실 주입

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

Research

JoVE Journal

Biology

21.1K 조회수.  Whitehead Institute for Biochemical Research. 신경 튜브 형성 후, neuroepithelium은 수축과 튜브가 배아 두뇌 심실을 형성하기 위해 배아 뇌척수 (eCSF)로 채우고 동안 주름. 우리는 더 나은 라이브 배아에서 neuroepithelial 모양 대조적으로 액체 채워진 공간을 시각화이 뇌실 주입 기술을 개발했습니다.

비디오: Zebrafish Brain Ventricle Injection

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

공촛점 현미경으로 Zebrafish 배아 뇌 영상 라이브

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

연구

생물학

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 ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

의 이식 GFP - 표현 키메라 Zebrafish의 태아의 망막과 두뇌 개발의 라이브 영상에 대한 Blastomeres을

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

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, ...

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

사출 및 Electroporation에 의해 Zebrafish Otocyst으로 MIF Morpholinos 직접 배달 내이 (内耳)의 개발 미치는 영향

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the ...

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 ...

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 ...

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

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to employ reverse genetics have not yet been adequately developed to assess gene function in regeneration or tissue homeostasis during larval stages after zebrafish embryogenesis, and several tissues within the zebrafish larva are difficult to target. Intraventricular injections of gene-specific morpholinos offer an alternative method for the current inability to genomically target zebrafish genes in a temporally controlled manner at these stages. This method allows for complete dispersion and subsequent incorporation of the morpholino into various tissues throughout the body, including structures that were formerly impossible to reach such as those in the larval caudal fin, a structure often used to noninvasively research tissue regeneration. Several genes activated during larval finfold regeneration are also present in regenerating adult vertebrate tissues, so the larva is a useful model to understand regeneration in adults. This morpholino dispersion method allows for the quick and easy identification of genes required for the regeneration of larval tissues as well as other physiological phenomena regulating tissue homeostasis after embryogenesis. Therefore, this delivery method provides a currently needed strategy for temporal control to the evaluation of gene function after embryogenesis.&#160;

An adaptable reverse genetic method for zebrafish to assess gene function during later stages of development and physiological homeostasis such as tissue regeneration using intraventricular injections of gene-specific morpholinos.

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 ...

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 ...