Name: visualization of cellular electrical activity in zebrafish early embryos and tumors
Uploaded: 2018-04-25T14:00:00
Description: Watch this Scientific Journal Video about Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors at JoVE.com

Forskningsarbeid i denne publikasjonen ble støttet av National Institute of General Medical Sciences av National Institutes of Health under prisen tall R35GM124913, Purdue University PI4D insentivprogram og PVM interne konkurransedyktig Grunnforskning midler programmet. Innholdet er ansvar forfattere og representerer ikke nødvendigvis den offisielle synet av finansiering agenter. Vi takker Koichi Kawakami for den Tol2 konstruksjonen, Michael Lin for den ASAP1 konstruksjonen, og Leonard Zon for ubi arrangøren konstruere gjennom Addgene.

Sebrafisk ligger i en AAALAC-godkjent dyr anlegget, og alle eksperimentene ble utført i henhold til protokoller godkjent av Purdue Animal Care og bruk Committee (PACUC).
1. Tol2 Transposon plasmider konstruere forberedelse
Merk: Tol2, en transposon som ble oppdaget i medaka fisk, er mye brukt i sebrafisk forskning samfunnet8,9. Det har vært vellykket vedtatt Gateway områdespesifikke rekombinasjon-basert klo...

<ol>
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Selv om de cellular og vev nivå elektriske aktivitetene under embryonale utvikling og menneskelig sykdom ble oppdaget for lenge siden, fortsatt i vivo dynamisk elektrisk endringene og deres biologiske roller hovedsakelig ubekjent. En av de store utfordringene er å visualisere og kvantifisere elektrisk endringene. Oppdateringen klemme teknologi er et gjennombrudd for sporing enkeltceller, men sin søknad til virveldyr embryoer er begrenset fordi består av mange celler. De gjeldende kjemiske spenning fargestoff...

Bioelectricity refererer til endogene elektrisk signalering formidlet av ionekanaler og pumper ligger på cellemembranen1. Ioniske utveksling mobilnettet membranen og kombinert elektrisk potensielle og nåværende endringene, er avgjørende for signalering prosesser av nervøs neuronal og muskel celler. I tillegg har bioelectricity og ion graderinger en rekke andre viktige biologiske funksjoner inkludert energilagring, biosyntesen og metabolitten transport. Bioelektrisk signalering ble også oppdaget en regulerende embryonale mønster formasjonen, som kroppen akser, cellen syklus og celle differensiering1. Derfor er det avgjørende for å forstå mange menneskelige medfødte sykdommer som følge av mis regulering av denne typen signalering. Selv om oppdateringen klemme har vært mye brukt for opptak enkeltceller, er det fortsatt langt fra ideell for samtidig overvåking av flere celler under embryonale utviklingen i vivo. Videre er spenning følsom små molekyler heller ikke perfekt for programmer i vivo sine særegenheter, følsomhet og toksisitet.
Etableringen av en rekke genetisk kodet fluorescerende spenning indikatorer (GEVIs) tilbyr en ny mekanisme for å overkomme dette problemet, og muliggjør enkel bruk å studere embryonale utvikling, selv om de var opprinnelig ment for overvåking nevrale 2,3i cellene. En av de tilgjengelige GEVIs er akselerert Sensor av handlingen potensialer 1 (ASAP1)4. Det består av en ekstracellulære løkke av et spenning-sensing domene spenning følsom fosfatase og et sirkulært permuted grønne fluorescerende protein. Derfor ASAP1 lar visualisering av mobilnettet elektrisk mulige endringer (polarisering: lys grønn; depolarization: mørk grønn). ASAP1 har 2 ms-og-på kinetics og kan spore subthreshold potensielle endre4. Dermed gir genetisk verktøyet et nytt nivå av effekt i real-time bioelectric overvåking i levende celler. Videre forståelse av rollene til bioelectricity i embryonale utvikling og mange menneskelige sykdommer som kreft, vil kaste nytt lys over de underliggende mekanismene som er avgjørende for behandling og forebygging.
Sebrafisk har vist en kraftig dyremodell utviklingsbiologi og menneskelige sykdommer, inkludert kreft5,6. De deler 70% orthologous gener med mennesker, og de har lignende virveldyr biologi7. Sebrafisk gir relativt enkel pleie, en stor clutch størrelse på egg, medgjørlig genetikk, lett transgenesis og gjennomsiktig eksterne embryonale utvikling, som gjør dem et bedre system for i vivo tenkelig5,6. Med en stor kilde til mutant fisk linjene allerede finnes og et fullt sekvensert genom, vil sebrafisk gi en relativt ubegrenset rekke vitenskapelige funn.
Undersøke i vivo sanntid elektriske aktiviteten til cellene, tar vi nytte av sebrafisk modell systemet og ASAP1. I dette papiret, vi beskriver hvordan å innlemme fluorescerende spenning biosensor ASAP1 i sebrafisk genomet bruker Tol2 transposon transgenesis, og visualisere mobilnettet elektrisk aktivitet under embryonale utviklingen, fisk larver bevegelse, og i live svulst .

<table>
	<tbody>
		<tr>
			<td>14mL cell culture tubes</td>
			<td>VWR</td>
			<td>60818-725</td>
			<td>E.Coli culture</td>
		</tr>
		<tr>
			<td>Agarose electrophoresis tank</td>
			<td>Thermo Scientific</td>
			<td>Owl B2</td>
			<td>DNA eletrophoresis</td>
		</tr>
		<tr>
			<td>Agarose RA</td>
			<td>Amresco</td>
			<td>N605-500G</td>
			<td>For making the injection gels</td>
		</tr>
		<tr>
			<td>Attb1-ASAP1-F primer</td>
			<td>IDT DNA</td>
			<td>GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATGGAGACGACTGTGAGGTATGAACA</td>
			<td>ASAP1 coding region amplification for subcloning</td>
		</tr>
		<tr>
			<td>Attb2-ASAP1-R primer</td>
			<td>IDT DNA</td>
			<td>GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAGGTTACCACTTCAAGTTGTTTCTTCTGTGAAGCCA</td>
			<td>ASAP1 coding region amplification for subcloning</td>
		</tr>
		<tr>
			<td>Bright field dissection scope</td>
			<td>Nikon</td>
			<td>SMZ 745</td>
			<td>Dechorionation, microinjection, mounting</td>
		</tr>
		<tr>
			<td>Color camera</td>
			<td>Zeiss</td>
			<td>AxioCam MRc</td>
			<td>Fish embryo image recording</td>
		</tr>
		<tr>
			<td>Concave slide</td>
			<td>VWR</td>
			<td>48336-001</td>
			<td>For holding fish embryos during imaging process</td>
		</tr>
		<tr>
			<td>Disposable transfer pipette 3.4 ml</td>
			<td>Thermo Scientific</td>
			<td>13-711-9AM</td>
			<td>Fish embryos and water transfer</td>
		</tr>
		<tr>
			<td>Endonuclease enzyme, Not I</td>
			<td>NEB</td>
			<td>R0189L</td>
			<td>For linearizing plasmid DNA</td>
		</tr>
		<tr>
			<td>Epifuorescent compound scope</td>
			<td>Zeiss</td>
			<td>Axio Imager.A2</td>
			<td>Fish embryo imaging</td>
		</tr>
		<tr>
			<td>Epifuorescent stereo dissection scope</td>
			<td>Zeiss</td>
			<td>Stereo Discovery.V12</td>
			<td>Fish embryo imaging</td>
		</tr>
		<tr>
			<td>Fluorescent light source</td>
			<td>Lumen dynamics</td>
			<td>X-cite seris 120</td>
			<td>Light source for fluorescence microscopes</td>
		</tr>
		<tr>
			<td>Forceps #5</td>
			<td>WPI</td>
			<td>500342</td>
			<td>Dechorionation and needle breaking</td>
		</tr>
		<tr>
			<td>Gateway BP Clonase II Enzyme mix</td>
			<td>Thermo Scientific</td>
			<td>11789020</td>
			<td>Gateway BP recombination cloning</td>
		</tr>
		<tr>
			<td>Gateway LR Clonase II Plus enzyme</td>
			<td>Thermo Scientific</td>
			<td>12538120</td>
			<td>Gateway LR recombination cloning</td>
		</tr>
		<tr>
			<td>Gel DNA Recovery Kit</td>
			<td>Zymo Research</td>
			<td>D4002</td>
			<td>DNA gel purification</td>
		</tr>
		<tr>
			<td>Loading tip</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td>For loading injection solution into capilary needles</td>
		</tr>
		<tr>
			<td>Methylcellulose (1600cPs)</td>
			<td>Alfa Aesar</td>
			<td>43146</td>
			<td>Fish embryo mounting</td>
		</tr>
		<tr>
			<td>Methylene blue</td>
			<td>Sigma-Aldrich</td>
			<td>M9140</td>
			<td>Suppresses fungal outbreaks in Petri dishes</td>
		</tr>
		<tr>
			<td>Microinjection mold</td>
			<td>Adaptive Science Tools</td>
			<td>TU-1</td>
			<td>To prepare agaorse mold tray for holding fish embryos during injection</td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>WPI</td>
			<td>Pneumatic Picopump PV820</td>
			<td>Microinjection injector</td>
		</tr>
		<tr>
			<td>Micro-manipulator</td>
			<td>WPI</td>
			<td>Microinjector mm33 rechts</td>
			<td>Microinjection operation</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter instrument</td>
			<td>P-1000</td>
			<td>For preparing capillary needle</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Amresco</td>
			<td>J217-500ml</td>
			<td>For calibrating injection volume</td>
		</tr>
		<tr>
			<td>mMESSAGE mMACHINE SP6 Transcription Kit</td>
			<td>Thermo Scientific</td>
			<td>AM1340</td>
			<td>mRNA in vitro transcription</td>
		</tr>
		<tr>
			<td>Monocolor camera</td>
			<td>Zeiss</td>
			<td>AxioCam MRm</td>
			<td>Fish embryo image recording</td>
		</tr>
		<tr>
			<td>Plasmid Miniprep Kit</td>
			<td>Zymo Research</td>
			<td>D4020</td>
			<td>Prepare small amount of plasmid DNA</td>
		</tr>
		<tr>
			<td>Plastic Petri dishes</td>
			<td>VWR</td>
			<td>25384-088</td>
			<td>For holding fish or fish embryos during imaging process</td>
		</tr>
		<tr>
			<td>RNA Clean &#38; Concentrator-5</td>
			<td>Zymo Research</td>
			<td>R1015</td>
			<td>mRNA cleaning after in vitro transcription</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>NanoDrop 2000</td>
			<td>For measuring DNA and RNA concentrations</td>
		</tr>
		<tr>
			<td>Stage Micrometer</td>
			<td>Am Scope</td>
			<td>MR100</td>
			<td>Microinjection volume calibration</td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>Bio-Rad</td>
			<td>T100</td>
			<td>DNA amplification for gene cloning</td>
		</tr>
		<tr>
			<td>Thin wall glass capillaries</td>
			<td>WPI</td>
			<td>TW100F-4</td>
			<td>Raw glass for making cappilary needle</td>
		</tr>
		<tr>
			<td>Tol2-exL1 primer</td>
			<td>IDT DNA</td>
			<td>GCACAACACCAGAAATGCCCTC</td>
			<td>Tol2 excise assay</td>
		</tr>
		<tr>
			<td>Tol2-exR primer</td>
			<td>IDT DNA</td>
			<td>ACCCTCACTAAAGGGAACAAAAG</td>
			<td>Tol2 excise assay</td>
		</tr>
		<tr>
			<td>TOP10 Chemically Competent E. coli</td>
			<td>Thermo Scientific</td>
			<td>C404006</td>
			<td>Used for transformation during gene cloning</td>
		</tr>
		<tr>
			<td>Tricaine mesylate</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
			<td>For anesthetizing fish or fish embryos</td>
		</tr>
		<tr>
			<td>UV trans-illuminator 302nm</td>
			<td>UVP</td>
			<td>M-20V</td>
			<td>DNA visualization</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Thermo Scientific</td>
			<td>2853</td>
			<td>For transformation process of gene cloning</td>
		</tr>
	</tbody>
</table>

visualization of cellular electrical activity in zebrafish early embryos and tumors

Bioelectricity, endogen elektrisk signalering formidlet av ionekanaler og pumper på cellemembranen, spiller viktige roller i signalering prosesser av nervøs neuronal og muskel celler og mange andre biologiske prosesser, som embryonale utviklingsmessige mønstre. Men er det behov for i vivo elektrisk aktivitet overvåking i virveldyr embryogenesis. Fremskritt i genetisk kodet fluorescerende spenning indikatorer (GEVIs) har gjort det mulig å gi en løsning på denne utfordringen. Her beskriver vi hvordan du oppretter en transgene spenning indikator sebrafisk bruker indikatoren etablerte spenning, ASAP1 (akselerert Sensor av handlingen potensialer 1), som et eksempel. Tol2 kit og en allestedsnærværende sebrafisk promoter, ubi, ble valgt i denne studien. Vi har også forklare prosessene av Gateway områdespesifikke kloning, Tol2 transposon-baserte sebrafisk transgenesis og avbildingsprosessen for tidlig stadium fisk embryoer og fisk svulster bruk av vanlig epifluorescent mikroskop. Bruker denne fisken linjen, fant vi at det er mobilnettet strømstyrken endringer under sebrafisk embryogenesis og fisk larver bevegelse. Videre ble det observert at i noen sebrafisk ondartet perifere nerve skjede svulster, svulst cellene var generelt polarisert forhold til de omliggende normalt vev.

I en vellykket injeksjon, mer enn 50% injisert fisk embryo vises noen grad av grønne fluorescens i somatiske celler, og de fleste av dem vil være positiv ved Tol2 transposon avgiftsdirektoratet analysen (figur 2). Etter 2-4 generasjoner av krysse ut med wildtype fisk (inntil fluorescerende fisken nå 50%, forventet Mendelian forholdet), ble transgene fisken brukt for bildebehandling eksperimentet spore cellemembranen potensialer under embryonale utviklingen...

Watch this Scientific Journal Video about Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors at JoVE.com

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Visualisering av mobilnettet elektrisk aktivitet i sebrafisk tidlig embryo og svulster

Her viser vi å lage en mobilnettet strømstyrken reporter sebrafisk linje for å visualisere embryonale utviklingen, bevegelse, og fisk tumor celler i vivo.

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Research

JoVE Journal

Developmental Biology

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Måling Protein Stabilitet på Living Sebrafisk embryo med fluoresens Decay Etter Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

Mechanical Vessel Injury in Zebrafish Embryos

Mekanisk Vessel Injury i sebrafisk embryo

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Taps og Gain-of-funksjon tilnærming for å undersøke Tidlig Cell Fate Determinanter i Før implantasjon Mouse Embryoer

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Transkriptom profilering av<em&gt; In-Vivo</em&gt; Produserte Bovine Pre-implantasjon Embryoet ved hjelp av to-farge microarray plattform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

Isolering og karakterisering av enkeltceller fra sebrafisk embryo

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

Biosensing Motor Neuron Membran Potensial i Live Zebrafish Embryoer

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Induksjon av hypoksi i levende frosk og sebrafiskembryoer

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Flere Foton tid forfalle Imaging se utvikling i sanntid: visualisering av migrere Neural Crest celler i sebrafisk embryo

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

Visualisere Node og Notochordal Plate i Gastrulating Mouse embryoer skanning elektronmikroskop og hele Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Hele Immunohistokjemi i sebrafiskembryoer og larver

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...