Name: microstructured devices for optimized microinjection and imaging of zebrafish larvae
Uploaded: 2017-12-08T13:00:00
Description: Watch this Scientific Journal Video about Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae at JoVE.com

Forfatterne vil gerne takke David Langenau for generøst giver akvarium rum; Eric Stone, John C. Moore og Qin Tang for hjælpe med zebrafisk vedligeholdelse og reagenser, og Anne Robertson og Elliott Hagedorn fra Leonard Zon lab for fremskaffelse af zebrafisk stamme bruges her. De vil også gerne takke Octavio Hurtado for rådgivning om photolithographic teknikker. FE blev finansieret af stipendier fra Shriner's Hospital for børn og den amerikanske australske sammenslutning. Dette arbejde blev finansieret af NIH give GM92804.

Mikroinjektion af larver var godkendt af Massachusetts General Hospital Underudvalget om forskning Animal Care under protokol 2011N000127.
1. enhed fabrikation
Bemærk: Alle computer bistået tegning (CAD) filer, der bruges til at designe fotolitografi masker beskrevet her (figur 1) er tilgængelig for download. Se tabel af materialer til links.
<ol><li>Fremstil master mold wafer i en klasse 1.000 rene rum ved hjælp af standardteknik...

<ol>
	<li>Lieschke, G. J., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+disease:+zebrafish+swim+into+view.">Animal models of human disease: zebrafish swim into view.</a> Nat Rev Genet. 8 (5), 353-367 (2007).</li><li>Dang, M., Fogley, R., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+Novel+Cancer+Therapies+Using+Chemical+Genetics+and+Zebrafish.">Identifying Novel Cancer Therapies Using Chemical Genetics and Zebrafish.</a> Adv Exp Med Biol. 916, 103-124 (2016).</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>Benard, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+of+zebrafish+embryos+with+intracellular+bacterial+pathogens.">Infection of zebrafish embryos with intracellular bacterial pathogens.</a> J Vis Exp. (61), (2012).</li><li>Ellett, F., Irimia, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microstructured+Surface+Arrays+for+Injection+of+Zebrafish+Larvae.">Microstructured Surface Arrays for Injection of Zebrafish Larvae.</a> Zebrafish. 14 (2), 140-145 (2017).</li><li>Bischel, L. L., Mader, B. R., Green, J. M., Huttenlocher, A., Beebe, D. 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(119), (2017).</li><li>Shao, G., Wu, J., Cai, Z., Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+elastomeric+high-aspect-ratio+microstructures+using+polydimethylsiloxane+(PDMS)+double+casting+technique.">Fabrication of elastomeric high-aspect-ratio microstructures using polydimethylsiloxane (PDMS) double casting technique.</a> Sens Actuators A Phys. 178, 230-236 (2012).</li><li>Bhuiyan, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acinetobacter+baumannii+phenylacetic+acid+metabolism+influences+infection+outcome+through+a+direct+effect+on+neutrophil+chemotaxis.">Acinetobacter baumannii phenylacetic acid metabolism influences infection outcome through a direct effect on neutrophil chemotaxis.</a> Proc Natl Acad Sci U S A. 113 (34), 9599-9604 (2016).</li><li>Henry, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PhagoSight:+an+open-source+MATLAB&#174;+package+for+the+analysis+of+fluorescent+neutrophil+and+macrophage+migration+in+a+zebrafish+model.">PhagoSight: an open-source MATLAB&#174; package for the analysis of fluorescent neutrophil and macrophage migration in a zebrafish model.</a> PloS one. 8 (8), e72636 (2013).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <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 Dynam. 203 (3), 253-310 (1995).</li><li>Hemmil&#228;, S., Cauich-Rodr&#237;guez, J. V., Kreutzer, J., Kallio, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+simple,+and+cost-effective+treatments+to+achieve+long-term+hydrophilic+PDMS+surfaces.">Rapid, simple, and cost-effective treatments to achieve long-term hydrophilic PDMS surfaces.</a> Applied Surface Science. 258 (24), 9864-9875 (2012).</li><li>Masselink, W., Wong, J. C., Liu, B., Fu, J., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-cost+silicone+imaging+casts+for+zebrafish+embryos+and+larvae.">Low-cost silicone imaging casts for zebrafish embryos and larvae.</a> Zebrafish. 11 (1), 26-31 (2014).</li><li>Yang, F., Gao, C., Wang, P., Zhang, G. J., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish-on-a-chip:+microfluidics+for+zebrafish+research.">Fish-on-a-chip: microfluidics for zebrafish research.</a> Lab Chip. 16 (7), 1106-1125 (2016).</li><li>Wielhouwer, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+embryo+development+in+a+microfluidic+flow-through+system.">Zebrafish embryo development in a microfluidic flow-through system.</a> Lab Chip. 11 (10), 1815-1824 (2011).</li><li>Shen, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+student+team+in+a+University+of+Michigan+biomedical+engineering+design+course+constructs+a+microfluidic+bioreactor+for+studies+of+zebrafish+development.">A student team in a University of Michigan biomedical engineering design course constructs a microfluidic bioreactor for studies of zebrafish development.</a> Zebrafish. 6 (2), 201-213 (2009).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+on+a+chip:+a+novel+platform+for+real-time+monitoring+of+drug-induced+developmental+toxicity.">Zebrafish on a chip: a novel platform for real-time monitoring of drug-induced developmental toxicity.</a> PLoS One. 9 (4), e94792 (2014).</li><li>Akagi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniaturized+embryo+array+for+automated+trapping,+immobilization+and+microperfusion+of+zebrafish+embryos.">Miniaturized embryo array for automated trapping, immobilization and microperfusion of zebrafish embryos.</a> PLoS One. 7 (5), e36630 (2012).</li><li>Akagi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish+on+chips:+Microfluidic+living+embryo+array+for+accelerated+in+vivo+angiogenesis+assays.">Fish on chips: Microfluidic living embryo array for accelerated in vivo angiogenesis assays.</a> Sens Actuators B Chem. 189, 11-20 (2013).</li><li>Lin, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+mapping+of+brain-wide+activity+in+awake+and+drug-responsive+vertebrates.">High-throughput mapping of brain-wide activity in awake and drug-responsive vertebrates.</a> Lab Chip. 15 (3), 680-689 (2015).</li><li>Noori, A., Selvaganapathy, P. 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Her, vi beskriver brugen af hjælpemidler vi for nylig udviklet for at lette 2 dpf zebrafisk mikroinjektion5, og indføre en simpel Agarosen-fri monteringsudstyret til praktisk billeddannelse af embryoner. Disse værktøjer fremhæve nytten af photolithographic teknikker til fabrikation af enheder, der er nyttige for zebrafisk teknikker.
Vi har fundet MSA enheder særlig nyttig for indsprøjtning af celler eller partikler tilbøjelige til sammenlægning inden for mikroi...

Zebrafisk er dukket op som en kraftfuld model for mange felter, fra studier af grundlæggende udviklingsmæssige biologi til store genetiske og kemiske skærme1,2. Rutinemæssig genetiske manipulationer, som genet overekspression, knockdown, CRISPR/Cas9 mutagenese og transgenese stole på mikroinjektion af genetisk materiale i én celle zygote, som har ført til udviklingen af enkle, let at bruge, kommercielt tilgængelige værktøjer til at orientere og stabilisere æg til injektion3. Andre tiltag, såsom transplantation og infektion, kræver ofte mikroinjektion i senere fase embryoner og larver ved hjælp af større sporvidde kapillær nåle4. Men brugen af større sporvidde nåle præsenterer store tekniske udfordringer, som det er mere vanskeligt at trænge målvæv uden at skubbe eller rullende embryonet. Under disse betingelser, kan at opnå passende vand spændinger skal stabilisere embryonet, mens undgå tørring under proceduren, der er vanskelige og embryoner ikke være ideelt orienteret til injektion i målvæv.
Efter mikroinjektion er det ofte nyttigt at screene injiceres embryoner til at vælge dem, der har været succesfuldt injiceres, og til at fange billeder af det oprindelige tidspunkt. For at imødegå disse udfordringer, har vi udviklet en række microstructured enheder, der bidrager til at stabilisere 2 dpf embryoner i forskellige retningslinjer for mikroinjektion5, såvel for hurtig image-baseret screening efter injektion.
For at opnå tilstrækkelig strukturelle opløsning i disse enheder, udnyttede vi photolithographic teknikker. Almindeligt anvendt i mikroelektroniske industrier og mere for nylig ekstrapoleret til mikrofluid fabrikation, kan disse tilgange opnå vertikale strukturer spænder fra 1-1.000 µm, en skala, der er velegnet til manipulering af zebrafisk embryoner og larver. Alle enheder var opdigtet benytter Polydimethylsiloxan (PDMS), som er billige, fysisk robust, biologisk inaktivt og gennemsigtig.
Microstructured overflade arrays (MSAs) var formateret som blokke af PDMS med en mønstret top overflade, svarer til de enkle kanaler i Agarosen blokke bruges normalt til æg mikroinjektion. For efter injektion screening, kan 6 billedenheder være klædt i en standard glas-bund 6-godt plade. Disse enheder er designet til nem pålæsning af embryoner, mens den losning procedure bekvemt tillader redning af specifikke embryoner, lette image-baseret screening tilgange i en mere brugervenlig måde end disse enheder tidligere udviklet af de Beebe laboratorium6.

<table>
	<tbody>
		<tr>
			<td>Dow Corning Sylgard 184&#160; Polydimethylsiloxane (PDMS)&#160;</td>
			<td>Ellsworth Adhsives</td>
			<td>184 SIL ELAST KIT 0.5KG</td>
			<td>For casting the devices. Kit includes PDMS monomer and Initiator</td>
		</tr>
		<tr>
			<td>Low gelling temperature agarose</td>
			<td>Sigma Aldrich</td>
			<td>A9414-10G</td>
			<td>For casting agarose devices</td>
		</tr>
		<tr>
			<td>PFDTS silane</td>
			<td>Sigma Aldrich</td>
			<td>448931-10G</td>
			<td>For casting of negative PDMS molds</td>
		</tr>
		<tr>
			<td>Tricaine (MS-222)</td>
			<td>Sigma Aldrich</td>
			<td>E10521-10G</td>
			<td>To anesthetize&#160; zebrafish&#160;</td>
		</tr>
		<tr>
			<td>Rhodamine Dextran 70,000 Da</td>
			<td>ThermoFisher</td>
			<td>D1818</td>
			<td>To trace microinjections</td>
		</tr>
		<tr>
			<td>Leukotriene B4 (LTB4)</td>
			<td>Cayman Chemicals</td>
			<td>20110</td>
			<td>Neutrophil chemoattractant</td>
		</tr>
		<tr>
			<td>N-Formylmethionine-leucyl-phenylalanine (fMLP)</td>
			<td>Sigma Aldrich</td>
			<td>F3506-50MG</td>
			<td>Neutrophil chemoattractant</td>
		</tr>
		<tr>
			<td>15 cm Petri dish</td>
			<td>Fisher scientific</td>
			<td>08-757-148</td>
			<td>For Casting from the master wafer</td>
		</tr>
		<tr>
			<td>Glass-bottom 6-well plates</td>
			<td>MatTek</td>
			<td>P06G-0-20-F</td>
			<td>For imaging devices</td>
		</tr>
		<tr>
			<td>Borosilicate glass microcapillaries</td>
			<td>World Scientific Instruments</td>
			<td>TW-100-4</td>
			<td>For microinjection needles</td>
		</tr>
		<tr>
			<td>Transfer pipettes</td>
			<td>Sigma Aldrich</td>
			<td>Z350796</td>
			<td>For transferring zebrafish embryos</td>
		</tr>
		<tr>
			<td>Microloader tips</td>
			<td>Fisher scientific</td>
			<td>E5242956003</td>
			<td>For loading the microinjection needles</td>
		</tr>
		<tr>
			<td>Harris Uni-Core 1.5 mm punch</td>
			<td>Ted Pella Inc.</td>
			<td>15111-15</td>
			<td>To punch ports in PDMS imaging devices</td>
		</tr>
		<tr>
			<td>No.&#160;11 Scalpel</td>
			<td>Fine Science Tools</td>
			<td>10011-00</td>
			<td>For cutting PDMS&#160;</td>
		</tr>
		<tr>
			<td>Dumont No. 5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-10</td>
			<td>For dechorionating embryos and breaking microinjection needle tips</td>
		</tr>
		<tr>
			<td>Marzhauser Micromanipulator</td>
			<td>ASI&#160;</td>
			<td>MM33-R</td>
			<td>For manipulating microinjection needle</td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td>MSC</td>
			<td>SPI - 87242624</td>
			<td>For mounting micromanipulator</td>
		</tr>
		<tr>
			<td>MPPI-3 Picopump controller</td>
			<td>ASI</td>
			<td>MPPI-3</td>
			<td>To control microinjection volume and timing</td>
		</tr>
		<tr>
			<td>EVOS inverted fluorescent microscope</td>
			<td>ThermoFisher</td>
			<td>EVOS FL</td>
			<td>To image injected embryos</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Nikon</td>
			<td>SMZ745</td>
			<td>For visualizing microinjecion</td>
		</tr>
		<tr>
			<td>AutoCAD software</td>
			<td>Autodesk</td>
			<td></td>
			<td>Download AutoCAD files from: https://dx.doi.org/10.6084/m9.figshare.4282853 and on the ZFIN community protocols wiki page: https://wiki.zfin.org/display/prot/ZFIN+ Protocol+Wiki &#160;</td>
		</tr>
	</tbody>
</table>

microstructured devices for optimized microinjection and imaging of zebrafish larvae

Zebrafisk er dukket op som en kraftfuld model af forskellige menneskelige sygdomme og et nyttigt redskab for en voksende række eksperimentelle undersøgelser, der spænder over grundlæggende udviklingsmæssige biologi gennem til store genetiske og kemiske skærme. Men, mange eksperimenter, især dem med tilknytning til infektion og xenograft modeller, stole på mikroinjektion og billeddannelse af embryoner og larver, som er besværlige teknikker, der kræver dygtighed og ekspertise. For at forbedre præcision og gennemløb af nuværende mikroinjektion teknikker, vi udviklet en serie af microstructured enheder til at orientere og stabilisere zebrafisk embryoner på 2 dage post befrugtning (dpf) i ventrale, dorsale eller laterale orientering forud for den procedure. For at støtte i billeddannelse af embryoner, designet vi også en simpel enhed med kanaler, orient 4 zebrafisk sideværts parallelt mod et glas dækning slip. Sammen, demonstrere de værktøjer, vi præsenterer her effektiviteten af photolithographic metoder til at generere nyttige enheder for optimering af zebrafisk teknikker.

Metoden beskrevet her viser design (figur 1) og fabrikation af enheder til brug med 2 dpf zebrafisk, ved hjælp af photolithographic (figur 2) og soft-litografi (fig. 3) teknikker. Denne metode giver mulighed for hurtig test af mange design iterationer og ændringer, og ombygninger og optimering af mikrostrukturen dimensioner til brug med zebrafisk i andre faser af udvikling kan udvide deres ansøgni...

Watch this Scientific Journal Video about Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae at JoVE.com

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Mikroinjektion af zebrafisk embryoner og larver er en afgørende men udfordrende teknik, der anvendes i mange zebrafisk modeller. Her præsenterer vi et udvalg af individuel værktøjer til at støtte i stabiliseringen og orientering af zebrafisk for både mikroinjektion og billedbehandling.

Microstructured anordninger til optimeret mikroinjektion og billeddannelse af zebrafisk larver

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