체외의 수정 및 gynogenetic의 diploids에 대한 방법은 1970 년대와 1980 년대 오레곤 대학의 조지 Streisinger의 실험실에 챠라인 워커에 의해 zebrafish를 위해 개발되었습니다. 방법은 여기에 설명된 것은 Zebrafish 도서 3 전체에서 사용할 수 있습니다 챠라인의 프로토콜에서 변하지입니다. 체외의 수정 및 gynogenetic의 diploids에 대한 방법은 1970 년대와 1980 년대 오레곤 대학의 조지 Streisinger의 실험실에 챠라인 워커에 의해 zebrafish를 위해 개발되었습니다. 방법은 여기에 설명된 것은 Zebrafish 도서 3 전체에서 사용할 수 있습니다 챠라인의 프로토콜에서 변하지입니다.

초기 압력의 개요 참고 사항 : tricaine, 생선 물 행크스 호수로이 프로토콜에서 사용되는 시약의 많은 요리법 Zebrafish 도서 3 조 (의 10 장에서 사용할 수있는 온라인 <a href="http://zfin.org/zf_info/zfbook/zfbk.html">http://zfin.org/zf_info/zfbook/zfbk.html</a> ) <ol><li> UV inactivated 정자를 사용하여 체외의 수정에 의해 배아를 생성합니다. </li><li> 즉시 계란 물을 브림에 가득찬 유리관 (플라스틱 스냅 캡 포함)로 수정된 달걀을 전송합니다. 라텍스 고무의 얇은 시트로 유리병의 열린 끝을 커버와 고무 위에 유리병에 상단에 (구멍이 절단되어있는로) 스냅인. </li><li> 액압 프레스의 병을 놓고 1.4 분 배입니다 8,000 PSI (많은 프레스는 2 인치 직경 피스톤에 끼쳤다 압력을 읽을 보정되며, 그 결과가 PSI로 변환해야합니다)에 압력을 올리기 </li><li> 수정 후 6 분 시작, 점차적으로 1 분 동안에 걸쳐 압력을 릴리스합니다. </li></ol> 상세 초기 압력 절차 <ol><li> 실험 전날 <ol style="list-style-type:lower-alpha;"><li> 늦은 오후에는 별도의 남성과 여성은 압착 수 있습니다. 그는 유전 물질을 공헌하지 않습니다 때문에 모든 중년 남성이 사용할 수 있습니다. 암컷은 뱃속에서 크고 뚱뚱해 보인다는 것입니다. 수컷은 노란색과 스프 라이 보일 것입니다. </li><li> 여성들이 쉽게 아침에 액세스할 수 있습니다 탱크를 들고 야간에 별도로 남성을 잡아. </li></ol></li><li> 실험의 아침 <ol style="list-style-type:lower-alpha;"><li> 행크스 솔루션을 준비, 나트륨 중탄산염를 추가합니다. </li><li> 생선 시설 지참 : 정자를 수집하기위한 얼음 양동이, 행크스, Tricaine, 타이머, 튜브. </li><li> 생선 물 Tricaine를 희석 </li></ol></li><li> 정자를 수집 <ol style="list-style-type:lower-alpha;"><li> 얼음 행크스 솔루션의 명확한 유리병을 준비합니다. 측정 ~ 남성 50 μl가 압착 수 있습니다. </li><li> tricaine에 집중하여 한 번에 3 또는 4 남자를 마취. 그들은 플라스틱 스푼으로 마취에서 해제하실 수 있습니다. </li><li> 생선 물에 씻어 종이 타월 (매우 중요 : 물 활성화의 정자)에 &quot;습기가 건조&quot;를 얼룩. </li><li> 에피 조명과 낮은 배율에서 stereomicroscope 아래 거품 홀더 물고기를 탑재합니다. 포셉와 골반 지느러미를 분리하여 하수의 개통에 microcapillary 위치를. </li><li> 부드럽게 그러나 단호하게 부드러운 (Millipore) 포셉와 물고기의 스트로크 측면. </li><li> 밀키 정자의 성기 구멍에서 나올으로 부드러운 흡입을 사용하여 microcapillary에 수집합니다. </li><li> 얼음처럼 차가운 행크의 호수에서 여러 남성에서 풀 정자. 50-10 남성의 정자는 수백 계란의 수정에 적합합니다. 추운 행크스의의 정자는 몇 시간 또는 며칠 동안 효율적으로 달걀을 비옥 것입니다. 흐린 정학을 할 수있을 정도로 충분한 수집 &quot;안구&quot;정자의 농도. </li></ol></li><li> 자외선은 정자를 inactivate <ol style="list-style-type:lower-alpha;"><li> 얼음 유리 페트리 접시의 바닥을 채우십시오. </li><li> 접시에 얼음 위에 watchglass 놔. </li><li> 파스퇴르 피펫을 사용하여 watchglass에 시험관에서 정자를 전송합니다. 그것으로 공기의 거품을 차지하지 않고 가능한 피펫으로 정자만큼 받아보실 수 있습니다. 또한 거품이 피펫에서 추방 그대로 watchglass에있는 정자하지 않습니다. 시계 유리에 얇은 심지어 레이어에 정자를 추방. 이 피펫을 삭제. </li><li> UV 크로스 링커에 페트리 접시를 놓습니다 </li><li> 이분에 대한 crosslinker 켭니다 </li><li> CLEAN 피펫을 사용하면, watchglass에서 정자를 제거하고 얼음 UV 정자와 장소를 표시 eppendorf 튜브에 전송할 수 있습니다. </li></ol></li><li> 에그 수집 및 체외은 수정 <ol style="list-style-type:lower-alpha;"><li> tricaine에서 한 번에 서너 여성을 마취. </li><li> 생선 물에 씻어 종이 타월에 &quot;습기가 건조&quot;를 얼룩. 초과 물이 알을 팽창하고 수정을 방지할 수 있습니다. </li><li> 35mm 플라스틱 페트리 접시와 습기 (서부 유럽 표준시되지 않음) 손가락으로 여자를 놓고, 부드럽게하지만 단단히 배꼽 누르십시오. 그녀가 알을 낳기 위해 준비하는 경우, 그들은 아주 쉽게 나올 것입니다. </li><li> 주걱으로 달걀을 모아서 물을 암컷을 반환합니다. (아래 참조) 여성하던 메뚜기 알이 너무 길고 흰 및 워터리 반면 좋은 계란은 노란, 반투명 색상입니다. 좋은 계란을 받고 보장하기 위해, 물고기 시설에서 오는 불빛 후 처음 2 시간 동안 그들을 수집합니다. 건조 아웃을 방지하기 위해 적용 달걀 보관하십시오. </li><li> , 달걀에 자외선 정자 정지의 30-50 μl를 추가 피펫 팁로 부드럽게 저어. </li><li> 즉시정 물고기 물 약 1 ML을 추가하고 타이머를 시작합니다. 이 시점에서 개발 왼쪽 달걀 haploids 것입니다. </li></ol></li><li> . 초기 압력 참고 : 단계 AG는 수정 후 90 초 내에 달성해야합니다! <ol style="list-style-type:lower-alpha;"><li> 계란은 물을 추가하여 활성화되는 순간에 타이머를 시작합니다 </li><li> 잠시 만요,하고 접시의 중앙에 더 많은 물, 소용돌이 계란을 추가합니다. </li><li> 차단과 광택 파스퇴르 피펫을 사용하여 압력 튜브에 수정된 달걀을 전송합니다. </li><li> 필요한 경우 그들은 거의 유리병의 입술에 물을 비드를 떠나, 넘쳐 수 있도록, 유리병에 더 많은 계란 물을 추가합니다. </li><li> 물의 구슬을 통해 고무 위로 플레이스와 고무 위에 플라스틱 뚜껑을 확보. </li><li> 리필 실린더에 더 많은 달걀 물을 추가 거꾸로 압력 실린더의 병을 (들)을 놓으십시오. 안전 실린더 위에 올려. </li><li> 플런저와 언론의 실린더를 놓고 8,000파운드 / 평방를 적용합니다. 인치 </li><li> 인증의 시간 후 6 분 (압력 4.5 분 총 시간)까지 언론에 실린더를 남겨주세요. </li><li> 활성화 후 6 분, 언론에서 실린더를 제거하고, 실린더의 튜브를 제거합니다. 조심스럽게 배아의 냉각을 방지하기 위해 튜브를 건조. </li><li> 실린더 및 리필 그 달걀 물로를 재설정하십시오. </li><li> 필요에 따라 부분적으로 계란 물을 그들을 작성하여 다음 튜브를 준비합니다. </li><li> 계란의 모든 일괄 처리에 대해 반복합니다. </li></ol></li></ol> 참고 : 실린더는 한 번에 세 튜브까지 개최되므로 이미 tricaine의 다른 암컷이 알을 주면 몇 분보고 알을 수정을 지연 할 수 있습니다. 이것은 한 번에 더 많은 일괄 처리하여 속도 것들을 도움이 될 것입니다. 또한, 한번 언론이 치료가 완료될 때까지 tricaine에서 더 많은 물고기를 넣어하지 사용되고 기억 해요. 마취를 유지하기 위해 물고기가 너무 오래 그들을 죽일 수 있도록합니다. 둘째, 당신이 언론이 사용되는 동안 물고기를 그만두라하고, 계란을하면, 그들은 언론을 다시 사용할 수있는 시간에 가능한 너무 건조 수 있습니다.

<ol>
	<li>Streisinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+analyses+and+gene-centromere+distances+in+zebrafish.">Segregation analyses and gene-centromere distances in zebrafish.</a> Genetics. 112 (2), 311-311 (1986).</li><li>Streisinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+clones+of+homozygous+diploid+zebra+fish+(Brachydanio+rerio).">Production of clones of homozygous diploid zebra fish (Brachydanio rerio).</a> Nature. 291 (5813), 293-293 (1981).</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 (Danio rerio). , (2000).</li><li>Johnson, S. L., Africa, D., Horne, S., Postlethwait, 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=Half-tetrad+analysis+in+zebrafish:+mapping+the+ros+mutation+and+the+centromere+of+linkage+group+I.">Half-tetrad analysis in zebrafish: mapping the ros mutation and the centromere of linkage group I.</a> Genetics. 139 (4), 1727-1727 (1995).</li><li>Beattie, C. E., Raible, D. W., Henion, P. D., Eisen, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+pressure+screens.">Early pressure screens.</a> Methods Cell Biol. 237 (71), 2575-25 (1999).</li><li>Johnson, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centromere-linkage+analysis+and+consolidation+of+the+zebrafish+genetic+map.">Centromere-linkage analysis and consolidation of the zebrafish genetic map.</a> Genetics. 142 (4), 1277-1277 (1996).</li><li>Trede, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+mutants+with+disrupted+early+T-cell+and+thymus+development+identified+in+early+pressure+screen.">Zebrafish mutants with disrupted early T-cell and thymus development identified in early pressure screen.</a> Dev Dyn. 237 (9), 2575-2575 (1999).</li></ol>

이 방법에 의해 만들어진 배아 사실 gynogenetic의 diploids되었는지 확인하는 것이 중요합니다. 컨트롤로 정상 diploid 달걀 (UV - 불활 성화없이 정자의 일부를 별도로 유지하여)와 haploid 배아 (EP 절차를 거치지 않고 UV 정자와 함께 수정된 계란의 클러치를 별도로 유지하여)의 클러치의 클러치를 생성합니다. 일일 게시물 수정의 haploid의 배아에서 짧은 몸 축, 불규칙한 두뇌와 체절의 형태 있습니다. Haploi...

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<td>Tricaine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Watch glass</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>French Press</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pressure cylinder</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Instant ocean</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur pipettes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Cut off the narrow end, fire polish</td>
</tr>
<tr>
<td>UV cross-linker</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>microcapillaries</td>
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making gynogenetic diploid zebrafish by early pressure

diploid의 eukaryotes에 Heterozygosity 종종 유전자 연구 성가신합니다. heterozygous 여성에서 직접 가능한 homozygous diploid의 자손을 생산 방법은 F1 mutagenized 여성이 가속 앞으로 유전 화면에 해로운 돌연변이 직접 검사를 할 수 있습니다. Streisinger 외.<sup&gt; 1,2</sup&gt;는 자외선 - inactivated 정자와 zebrafish 달걀을 활성화하고 물리 치료 (열 또는 압력)을 deploymerize microtubules를 사용하여 두 번째 meiotic 또는 첫 번째 접합자의 세포 분열 중 하나를 방지하여 gynogenetic (homozygous) diploid의 zebrafish를 만들기위한 방법을 설명했다. 빠른 시일 내에 수정 후 발생하는 &quot;초기 압력&quot;(EP) 메서드 블록 감수 분열 II. EP 방법도 섹스의 비옥한 성인 개발할 수 있습니다 가능한 배아의 높은 비율을 생산하고 있습니다. 방법은 세포의 감수 분열 I. Homozygous 변이가 centromeric loci 50 %와 telomeric loci에 대한 1 % 미만 사이에서 EP의 클러치에서 감지하는 동안 재조합하여 centromere에서 분리된 것을 제외한 모든 loci에 homozygous 아르 배아를 생성합니다. 이 방법은 Zebrafish 주소록에서 그대로 재현<sup&gt; 3</sup&gt;.

Watch this Scientific Journal Video about Making Gynogenetic Diploid Zebrafish by Early Pressure at JoVE.com

Making Gynogenetic Diploid Zebrafish by Early Pressure

이것은 바로 자외선 - inactivated 정자와 수정 후 두 번째 meiotic 부서 차단하여 gynogenetic의 diploid의 zebrafish의 배아 (단 유전적 기여 어머니의 유래 배아)를 생성하는 방법입니다. EP의 배아는 첫 번째 meiotic 부문 중 재조합에 의해 완전히 homozygous하지 않습니다, 그러나 그들은 재조합하여 centromere에서 분리되지 않은 모든 loci에 homozygous 있습니다.

초기 압력에 의해 Gynogenetic의 Diploid의 Zebrafish 만들기

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from ...

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Biology

13.4K 조회수.  University of Oregon. 이것은 바로 자외선 - inactivated 정자와 수정 후 두 번째 meiotic 부서 차단하여 gynogenetic의 diploid의 zebrafish의 배아 (단 유전적 기여 어머니의 유래 배아)를 생성하는 방법입니다. EP의 배아는 첫 번째 meiotic 부문 중 재조합에 의해 완전히 homozygous하지 않습니다, 그러나 그들은 재조합하여 centromere에서 분리되지 않은 모든 loci에 homozygous 있습니다.

비디오: Making Gynogenetic Diploid Zebrafish by Early Pressure

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

Brain Banking: Making the Most of your Research Specimens

Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given area of interest1. To achieve this goal 6-10 systematic sections should be probed covering the entire structure. Typically this involves processing 1/5 sections which leaves a significant amount of material unprocessed. In order to maximize the material, we propose an inexpensive method for preserving fixed tissue as part of a long-term storage research plan. As tissue is sliced and processed for the desired stain or antibody, alternate sections should be systematically placed in antigen preserve at -20&deg;C for future processing. Using 24-well plates, sections can be placed in order for future retrieval. Using this method, tissue can be stored and processed for immunohistochemistry over the course of years.

Brain banking and systematic sampling of biological material provides the basis for unbiased stereology and maximizes the potential data obtained from each specimen.

브레인 은행 : 귀하의 연구 표본의 대부분을 만들기

Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given area of interest1.  To ...

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 ...

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 &mu;m wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 &mu;m and ~40 &mu;m, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.

Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.

LAESI 질량 분석법에 의해 생물 학적 조직 및 Biofilms의 대기 압력 분자 이미징

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

대기압에서 질량 분석법에 의해 단일 ​​세포의 직접 분석

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

신진 대사 라벨링 및 Bioorthogonal 클릭 화학에 의해 Zebrafish 태아의 영상 Glycans

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

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 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

반수체 제브라 피쉬 배아의 생산에 의해<em&gt; 체외</em&gt; 시비

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

대변​​ 글루코 코르티코이드 분석 : equids는 비 침습적 부신 모니터링

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...