우리는 그들의 지원이 원고에 대한 도움 의견을 Barresi 연구소의 회원을 감사드립니다. 우리는 그의 지속적인 기술 지원을위한 알렉산더 워크맨뿐만 아니라 스미스 대학 Zebrafish의 식민지를 유지하는 그들의 도움 동물 관리 직원을 감사드립니다. 우리는 또한이 프로토콜의 filmin위한 현미경 장비를 대출에 대한 마이크 Hallacy, 루디 Rottenfusser, 그리고 칼 자이스 혈구 Microimaging 감사합니다. 이 작품은 NSF 자금 연구 부여, 0,615,594에 의해 지원되었다.

<ol>
	<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+guide+for+the+laboratory+use+of+zebrafish+(Danio.">A guide for the laboratory use of zebrafish (Danio.</a> The Zebrafish Book. , (2007).</li><li>N&#252;sslein-Volhard, C., Dahm, R. e. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+A+Practical+Approach.">Zebrafish A Practical Approach.</a> , (2002).</li><li>Graeden, E., 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=Live+imaging+of+the+zebrafish+embryonic+brain+by+confocal+microscopy.">Live imaging of the zebrafish embryonic brain by confocal microscopy.</a> J Vis Exp. (26), (2009).</li><li>Gilmour, D. T., Jessen, J. R., Lin, S., N&#252;sslein-Volhard, C., Dahm, R. e. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish,+a+practical+approach.">Zebrafish, a practical approach.</a> 1, 121-121 (2002).</li><li>Woo, K., Shih, J., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+maps+of+the+zebrafish+embryo.">Fate maps of the zebrafish embryo.</a> Curr Opin Genet Dev. 5 (4), 439-439 (1995).</li><li>Barresi, M. J., Hutson, L. D., Chien, C. B., Karlstrom, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+regulated+Slit+expression+determines+commissure+and+glial+cell+position+in+the+zebrafish+forebrain.">Hedgehog regulated Slit expression determines commissure and glial cell position in the zebrafish forebrain.</a> Development. 132 (16), 3643-3643 (2005).</li><li>Kozlowski, D. J., Murakami, T., Ho, R. K., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+cell+movement+and+tissue+patterning+in+the+zebrafish+embryo+revealed+by+fate+mapping+with+caged+fluorescein.">Regional cell movement and tissue patterning in the zebrafish embryo revealed by fate mapping with caged fluorescein.</a> Biochem Cell Biol. 75 (5), 551-551 (1997).</li><li>Halloran, M. C., Murakami, T., Ho, R. K., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-induced+gene+expression+in+specific+cells+of+transgenic+zebrafish.">Laser-induced gene expression in specific cells of transgenic zebrafish.</a> Development. 127 (9), 1953-1953 (2000).</li><li>Hardy, M. E., Ross, L. V., Chien, 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=Focal+gene+misexpression+in+zebrafish+embryos+induced+by+local+heat+shock+using+a+modified+soldering+iron.">Focal gene misexpression in zebrafish embryos induced by local heat shock using a modified soldering iron.</a> Dev Dyn. 236 (11), 3071-3071 (2007).</li></ol>

The authors have nothing to disclose.

키메라 배아를 생성하는 것은 여러 모델 시스템, 즉 과일 파리 (D.melanogaster), 웜 (C.elegans), zebrafish (D.rerio) 및 마우스 (M.musculus) 내에서 여러 연구 질문을 해결할 수있는 강력한 도구입니다. 우리는 zebrafish 모델 시스템 고유 비교적 쉽고 빠르고 다양한 방법으로 chimeras 생성에 적합한 것을 주장한다. zebrafish는 훨씬 접근 마우스 모델에 비해 만드는 어머니 외부 개발 척추입니다. Zebrafish 또한 세포 이식으로 embryological 조작을 단순화 파리 또는 웜보다 훨씬 큽니다. 또한, 유전자 변형 기술과 세포 이식 절차를 조합하는 능력은 유전자 기능화된 조직에 특정한 방식으로 조작할 수있는 가능한 실험의 큰 배열을 수 있습니다. 예를 들어, GFP + 또는 Rhodamine 분류 세포의 작은 클론은 종종 homozygous 유전자 변형 기증자의 분실 또는 일반적인 항체와 차동 subcellular 라벨에 의한 시각화하는 것은 불가능입니다 전체 셀 morphologies의 특성을 설정합니다. 또한, transgenically 태그 또는 찬란 채워진 세포를 사용하여, 우리는 시간이 지남에 라이브 zebrafish 배아에서 기증자 세포를 추적할 수 있습니다. 개별 세포의 Timelapse 이미징은 모든 유기체의 맥락에서 세포 행동의 소설 분석을 제공합니다. 우리 연구실은 열 충격의 사용은 특정 축삭지도 단서가 배양 온도 상승 (데이터가 표시되지 않음) 다음과 같은 기증자 세포 misexpressed 수있는 이러한 세포 transplantations와 유전자 변형 라인 inducible 결합되어 있습니다. 이 기술은 로컬 우리가 특정 단백질 개발에 영향을 미치는 방식을 볼 수있는 공간과 시간적 특정 방식에 관심 단백질을 misexpress하는 매우 강력하고 직접적인 방법입니다. 우리의 경우,이 기술은 commissures과 정중선 5 glial 세포의 위치에 슬릿 - 로보지도 단서의 기능 뒤에 우리의 지식을 확장할 수 있습니다. 같은 초점 자외선 uncaging 및화된 heatshock 도구 (레이저 또는 납땜 인두)과 같은 로컬 misexpressing 유전자에 다른 방법은 유전자 및 세포 7-9의 작은 클러스터에서 마커의 표현을 조작하는 다른 방법을 제공 할 수 있지만, 세포 이식은 확립 UV 레이저 또는 열 세포 이식보다 제어 실험적인 접근함으로써 유발하는 외상 무료입니다 분석의 최종 환경. 결론적으로, 우리는 neurodevelopmental 생물학 연구의 중요한 일부로 세포 이식 절차를 발견했다. 서로 다른 특성을 가진 세포의 클론을 생성하는 세포 행동, 유전자 기능 및 세포 자치의 근본적인 문제를 해결의 발달 생물학을위한 중요한 도구입니다.

<table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 이름 </td><td> 유형 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 구체적인 </td></tr><tr><td> 배양 접시 </td><td> 도구 </td><td> Fisherbrand </td><td> 08-757-13 </td><td> 100 X 15mm </td></tr><tr><td> 유리 바닥 문화 플레이트 </td><td> 도구 </td><td> MatTek </td><td> P35G - 1.5-2.0 - C </td><td> 35 X 15mm, 아니 1.5, Uncoated, 감마 조사 </td></tr><tr><td> 와이드 보어 유리 파스퇴르 pipets </td><td> 도구 </td><td> Fisherbrand </td><td> 63A - 53 - WT </td><td></td></tr><tr><td> 트러프 금​​형 유리 필라멘트 모세관 (필라멘트없이) 및 사출 바늘 (필라멘트와 함께) </td><td> 도구 </td><td> 세계 정밀 계측기 </td><td> 1B150F - 4 </td><td> 인치 4 (100mm), 1.5 / 0.84 OD / ID (㎜) </td></tr><tr><td> 이식 모세 혈관 (TransferTip) </td><td> 도구 </td><td> Eppendorf </td><td> 83000122-8 </td><td> 유형 IV, 멸균, INT. 15μm, 20도 구부 </td></tr><tr><td> 이식 형 </td><td> 도구 </td><td> 적응형 과학 도구 </td><td> PT - 1 </td><td> 개인 배아를 개최 150 삼각 divots </td></tr><tr><td> 아가로 오스, 나를 입력하십시오 </td><td> 시약 </td><td> 시그마 알드리치 </td><td> A6013 - 250G </td><td> 1.5 % 아가로 오스는 EM에서 만들어진 </td></tr><tr><td> (항생제) 엠브료 미디엄 </td><td> 시약 </td><td></td><td></td><td> 웨스 터, 2007을 참조하십시오 </td></tr><tr><td> 인산염 버퍼 </td><td> 시약 </td><td></td><td></td><td> 웨스 터, 2007을 참조하십시오 </td></tr><tr><td> 와치먼 집게 </td><td> 도구 </td><td> 파인 과학 도구 </td><td></td><td> 100 + mm 팁 (재미), 50mm 팁 </td></tr><tr><td> Rhodamine B Dextran </td><td> 리니지 추적기 염료 </td><td> 분자 프로브, Invitrogen </td><td> D1824 </td><td> MW 10,000 다, 그린 (570nm) 빛을 기대 라이신 - 고칠수 있고, 빨강 (590nm) 형광 염료 </td></tr><tr><td> 해부 현미경 </td><td> 현미경 </td><td> 올림푸스 </td><td> SZX7 </td><td></td></tr><tr><td> 형광 스테레오 현미경 </td><td> 현미경 </td><td> 자이스 혈구 </td><td> Lumar </td><td> 완전 자동화된 조이스틱 제어 (Sycop) </td></tr><tr><td> 이식 장치 </td><td> 도구 </td><td> Eppendorf 주식 회사 </td><td> AirTram </td><td></td></tr><tr><td> 자동 Micromanipulator </td><td> 도구 </td><td> Eppendorf 주식 회사 </td><td> TransferMan NK2 </td><td> 조이스틱 제어 </td></tr><tr><td> Micromanipulator </td><td> 도구 </td><td> 응용 과학 인 스트 루먼트 (주) </td><td> 00-42-101-0000 </td><td> MM33 오른쪽 </td></tr><tr><td> 백 압력 단위로 Microinjector </td><td> 도구 </td><td> 응용 과학 인 스트 루먼트 (주) </td><td> MPPI - 2 BPU </td><td></td></tr><tr><td> Flamming / 브라운 Micropipet 풀러 </td><td> 도구 </td><td> 서터 악기 회사 </td><td> 모델 P97 </td><td> 프로그램 : 열 550; 속도 200; 시간 / 지연 200; 힘 120 당기세요. </td></tr></tbody></table>

tissue targeted embryonic chimeras: zebrafish gastrula cell transplantation

세포 소설 환경에 배치하거나 더 큰 맥락에서 세포의 작은 그룹이 변경될 때 때 발달 생물학 분야의 특정 근본적인 질문에만 답변을 얻을 수 있습니다. 세포 하나가와 상호 작용하고 독특한 환경에서 동작하는 방법을 감상하는 것은 세포의 기능을 특성화하는 것이 필수적입니다. 세포를 둘러싼 특정 단백질 영향의 언어 misexpression은 단백질 개발 프로세스의 다양한 재생하는 역할을 통찰력 정보를 제공하는 방법 결정. 우리 연구실은 고유 genotypic 또는 phenotypic chimeras를 생성하는 고전적인 이식 기술과 유전자 접근 방법을 결합 zebrafish 모델 시스템을 사용합니다. 우리는 zebrafish의 forebrain의 commissures 형성하는 동안 신경 - glial 세포 상호 작용을 공부합니다. 이 비디오는 우리 연구소 astroglial diencephalon의 인구와 그들이 정중선을 교차으로 예상 axons에 영향을 미치는 구체적인지도 단서의 역할의 역할을 조사 수있는 방법을 설명합니다. 자신의 투명 zebrafish의 배아로 인해 이소성 셀 배치 또는화된 유전자 misexpression 이러한 유형의 이상적 모델입니다. 추적 이식 세포가 중요한 염료 또는 형광 단백질을 표현하는 유전자 변형 물고기 라인을 사용하여 수행할 수 있습니다. 이식을위한 중요한 염료의 추적과 기증자의 배아를 준비하는 방법을 우리는 gastrula는 다른 배아를 연출 하나에서 세포를 추출하고 이식하는 방법뿐만 아니라, 여기를 보여줍니다. 우리는 zebrafish 배아의 forebrain 내에 이소성 GFP 유전자 변형 세포 +를 보여주는 데이터를 제시하고 commissures을 forebrain에 관하여 이러한 세포의 위치를​​ 특징. 또한, 우리는 GFP 유전자 변형 + 호스트 배아에 이식 알렉사 594 레이블 셀 레이저 스캐닝 공촛점 timelapse 현미경을 보여줍니다. 이러한 데이터는 gastrula 개최 이식이 발달 생물학의 질문에 다양한 주소로 이소성 세포의 타겟 위치를 가능하게 증거를 제공합니다.

Watch this Scientific Journal Video about Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation at JoVE.com

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Zebrafish 세포 이식은 조직 특정 chimeras를 생성하는 유전 발생학의 조합이 가능합니다. 이 비디오는 gastrula 우리 연구실은 forebrain의 접합면의 형성 중에 astroglial 인구와 구체적인지도 단서의 역할을 조사하기 위해 허용해야 세포 transplantations를 개최 보여줍니다.

조직 타겟 배아 Chimeras : Zebrafish Gastrula 세포 이식

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

tissue-targeted-embryonic-chimeras-zebrafish-gastrula-cell

Research

JoVE Journal

Biology

19.1K 조회수.  Smith College. Zebrafish 세포 이식은 조직 특정 chimeras를 생성하는 유전 발생학의 조합이 가능합니다. 이 비디오는 gastrula 우리 연구실은 forebrain의 접합면의 형성 중에 astroglial 인구와 구체적인지도 단서의 역할을 조사하기 위해 허용해야 세포 transplantations를 개최 보여줍니다.

비디오: Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

성인 Zebrafish의 전체 신장 마로우의 이식

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

연구

생물학

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

굴지 공동 침강 분석, F - 굴지에 바인딩 단백질의 분석

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

이식에 의해 키메라 Zebrafish의 배아를 생성

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

Zebrafish 배아 눈 조직의 Microdissection

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps 3 so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.

Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. This video shows step-by-step procedures for how to manipulate medaka embryos, including dechorionation, mounting in agarose for imaging and cell transplantation for the production of chimeras. These procedures are essential to use medaka and zebrafish in a laboratory to take full advantage of their complementary features for the genetic dissection of vertebrate genome functions.

Chimeras의 생성에 대한 Medaka의 태아 및 세포 이식의 Dechorionation

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

트렌스 제닉 Zebrafish의 여러 가지 빛깔의 시간 저속 영상 : 타겟의 연결을 세포 절제에 의해 활성화 떠올리 망막 줄기 세포

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

에서 기본 셀 문화 Drosophila Gastrula의 태아

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

직접 성인 Zebrafish의 신장에 세포의 이식

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis1. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample2, 3. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. 
Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

Syngeneic Zebrafish의 제한 희석 세포 이식을 사용하여 종양 - 증식 세포의 주파수 Quantifying

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

Immunostaining of Dissected Zebrafish Embryonic Heart

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

해부 Zebrafish 배아 심장의 Immunostaining

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...