Name: in vivo imaging of transgenic gene expression in individual retinal progenitors in chimeric zebrafish embryos to study cell nonautonomous influences
Uploaded: 2017-03-22T15:00:00
Description: Watch this Scientific Journal Video about In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences at JoVE.c

Dette arbejde blev støttet af en ARC Decra til PRJ (DE120101311) og ved en Deutsche Forschungsgemeinschaft (DFG) forskningsbevilling til LP (PO 1440 / 1-1). Den australske regenerativ medicin Institute er støttet af midler fra delstatsregeringen i Victoria og det australske forbundsregering. Vi anerkender Dr. Jeremy Ng Chi Kei, der udført eksperimenter beskrives her som offentliggjort i Kei et al. , 2016. Vi er taknemmelige for bestemmelser i transgene fisk fra Prof. Higashijima og tak Profs. Turner og Rupp for levering af pCS2 + plasmidet og Dr. Wilkinson til at generere H2B-RFP og H2A-GFP konstruktioner. Vi takker FishCore facilitet personale (Monash University) til at tage sig af vores dyr.

Alle procedurer blev udført i overensstemmelse med bestemmelserne i den australske National Health og Medical Research Council regelsæt for pasning og anvendelse af dyr, og blev godkendt af de institutionelle etiske råd. 1. Udarbejdelse af Zebrafisk <ol><li> Voksen fisk parring <ol><li> Opsæt voksne fisk som par (eller to par) i passende størrelse ynglende tanke aftenen før anvendelse. </li><li> For at holde den kvindelige adskilt fra mandlige, bruge en deler, således at fisk at se,...

<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, 353-367 (2007).</li><li>Scholpp, S., Poggi, L., Zigman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+on+the+stage+-+spotlight+on+nervous+system+development+in+zebrafish:+EMBO+practical+course,+KIT,+Sept.+2013.">Brain on the stage - spotlight on nervous system development in zebrafish: EMBO practical course, KIT, Sept. 2013.</a> Neural Dev. 8, 23 (2013).</li><li>Cayouette, M., Poggi, L., Harris, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+in+the+vertebrate+retina.">Lineage in the vertebrate retina.</a> Trends Neurosci. 29, 563-570 (2006).</li><li>Poggi, L., Vitorino, M., Masai, I., Harris, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influences+on+neural+lineage+and+mode+of+division+in+the+zebrafish+retina+in+vivo.">Influences on neural lineage and mode of division in the zebrafish retina in vivo.</a> J Cell Biol. 171, 991-999 (2005).</li><li>Poggi, L., Zolessi, F. R., Harris, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+analysis+of+retinal+differentiation.">Time-lapse analysis of retinal differentiation.</a> Curr Opin Cell Biol. 17, 676-681 (2005).</li><li>Jusuf, P. R., 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=Biasing+amacrine+subtypes+in+the+Atoh7+lineage+through+expression+of+Barhl2.">Biasing amacrine subtypes in the Atoh7 lineage through expression of Barhl2.</a> J Neurosci. 32, 13929-13944 (2012).</li><li>Jusuf, P. R., 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=Origin+and+determination+of+inhibitory+cell+lineages+in+the+vertebrate+retina.">Origin and determination of inhibitory cell lineages in the vertebrate retina.</a> J Neurosci. 31, 2549-2562 (2011).</li><li>Jusuf, P., Harris, W. A., Poggi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+retinal+progenitor+lineages+in+developing+zebrafish+embryos.">Imaging retinal progenitor lineages in developing zebrafish embryos.</a> Cold Spring Harb Protoc. 2013, (2013).</li><li>Jusuf, P., Harris, W. A., Poggi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+transgenic+zebrafish+embryos+for+imaging+the+developing+retina.">Preparation of transgenic zebrafish embryos for imaging the developing retina.</a> Cold Spring Harb Protoc. 2013, (2013).</li><li>Vitorino, 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=Vsx2+in+the+zebrafish+retina:+restricted+lineages+through+derepression.">Vsx2 in the zebrafish retina: restricted lineages through derepression.</a> Neural Dev. 4, 14 (2009).</li><li>Reh, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+regulation+of+neuronal+production+in+the+larval+frog+retina.">Cell-specific regulation of neuronal production in the larval frog retina.</a> J Neurosci. 7, 3317-3324 (1987).</li><li>Reh, T. A., Tully, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+tyrosine+hydroxylase-containing+amacrine+cell+number+in+larval+frog+retina.">Regulation of tyrosine hydroxylase-containing amacrine cell number in larval frog retina.</a> Dev Biol. 114, 463-469 (1986).</li><li>Livesey, F. J., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertebrate+neural+cell-fate+determination:+lessons+from+the+retina.">Vertebrate neural cell-fate determination: lessons from the retina.</a> Nature reviews. Neuroscience. 2, 109-118 (2001).</li><li>Zolessi, F. R., Poggi, L., Wilkinson, C. J., Chien, C. B., Harris, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarization+and+orientation+of+retinal+ganglion+cells+in+vivo.">Polarization and orientation of retinal ganglion cells in vivo.</a> Neural Dev. 1, 2 (2006).</li><li>Kimura, Y., Okamura, Y., Higashijima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alx,+a+zebrafish+homolog+of+Chx10,+marks+ipsilateral+descending+excitatory+interneurons+that+participate+in+the+regulation+of+spinal+locomotor+circuits.">alx, a zebrafish homolog of Chx10, marks ipsilateral descending excitatory interneurons that participate in the regulation of spinal locomotor circuits.</a> J Neurosci. 26, 5684-5697 (2006).</li><li>Kimura, Y., Satou, C., Higashijima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=V2a+and+V2b+neurons+are+generated+by+the+final+divisions+of+pair-producing+progenitors+in+the+zebrafish+spinal+cord.">V2a and V2b neurons are generated by the final divisions of pair-producing progenitors in the zebrafish spinal cord.</a> Development. 135, 3001-3005 (2008).</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). , (2007).</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 Dyn. 203 (3), 253-310 (1995).</li><li>Kei, J. N., Dudczig, S., Currie, P. D., Jusuf, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feedback+from+each+retinal+neuron+population+drives+expression+of+subsequent+fate+determinant+genes+without+influencing+the+cell+cycle+exit+timing.">Feedback from each retinal neuron population drives expression of subsequent fate determinant genes without influencing the cell cycle exit timing.</a> J Comp Neurol. 524, 2553-2566 (2016).</li><li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+mRNA+and+morpholino+antisense+oligonucleotides+in+zebrafish+embryos.">Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos.</a> J Vis Exp. , (2009).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> J Vis Exp. , (2009).</li><li>Zou, J., Wei, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+GFP-expressing+blastomeres+for+live+imaging+of+retinal+and+brain+development+in+chimeric+zebrafish+embryos.">Transplantation of GFP-expressing blastomeres for live imaging of retinal and brain development in chimeric zebrafish embryos.</a> J Vis Exp. , (2010).</li><li>Kemp, H. A., Carmany-Rampey, A., Moens, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+chimeric+zebrafish+embryos+by+transplantation.">Generating chimeric zebrafish embryos by transplantation.</a> J Vis Exp. , (2009).</li><li>Schier, A. F., Talbot, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+genetics+of+axis+formation+in+zebrafish.">Molecular genetics of axis formation in zebrafish.</a> Annu Rev Genet. 39, 561-613 (2005).</li><li>De Luca, E., 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=ZebraBeat:+a+flexible+platform+for+the+analysis+of+the+cardiac+rate+in+zebrafish+embryos.">ZebraBeat: a flexible platform for the analysis of the cardiac rate in zebrafish embryos.</a> Sci Rep. 4, 4898 (2014).</li><li>Lin, J. W., 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=Differential+requirement+for+ptf1a+in+endocrine+and+exocrine+lineages+of+developing+zebrafish+pancreas.">Differential requirement for ptf1a in endocrine and exocrine lineages of developing zebrafish pancreas.</a> Dev Biol. 274, 491-503 (2004).</li><li>Hollyfield, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histogenesis+of+the+retina+in+the+killifish,+Fundulus+heteroclitus.">Histogenesis of the retina in the killifish, Fundulus heteroclitus.</a> J Comp Neurol. 144 (3), 373-380 (1972).</li><li>La Vail, M. M., Rapaport, D. H., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytogenesis+in+the+monkey+retina.">Cytogenesis in the monkey retina.</a> J Comp Neurol. 309 (1), 86-114 (1991).</li><li>Rapaport, D. H., Wong, L. L., Wood, E. D., Yasumura, D., LaVail, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+and+topography+of+cell+genesis+in+the+rat+retina.">Timing and topography of cell genesis in the rat retina.</a> J Comp Neurol. 474 (2), 304-324 (2004).</li><li>Sharma, S. C., Ungar, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histogenesis+of+the+goldfish+retina.">Histogenesis of the goldfish retina.</a> J Comp Neurol. 191 (3), 373-382 (1980).</li><li>Stiemke, M. M., Hollyfield, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+birthdays+in+Xenopus+laevis+retina.">Cell birthdays in Xenopus laevis retina.</a> Differentiation. 58 (3), 189-193 (1995).</li></ol>

Forståelse hvilket omfang naboceller påvirke timing af ekspressionen af ​​afgørende celleskæbne afgørende faktorer er afgørende, når der sigtes til effektivt instruere embryonale eller inducerede multipotente stamceller til at differentiere til en specifik post-mitotisk celletype eller endda mønstrede væv. Desuden undersøger disse molekylære begivenheder i udviklingslandene celler i det levende dyr derudover giver relevant dynamik (tidslige og rumlige) oplysninger om de særlige cellulære sammenhænge i ...

<html> Evnen til at visualisere vigtige udviklingsprocesser i en in vivo hvirveldyr har bidraget til at gøre zebrafisk en vigtig model til at studere normale og sygdomstilstande (revideret i 1, 2). Især den neurale nethinde er en tilgængelig del af det centrale nervesystem. Nethinden egner sig til nemt at udføre undersøgelser af neurogenese på grund af sin meget organiseret, men alligevel relativt enkle struktur, og dets højt konserverede neuron typer tværs hvirveldyr 3. Dynamics af cellulære adfærd, såsom spredning, cellecyklus exit, asymmetrisk celledeling, skæbne specifikation, differentiering og neurale kredsløb dannelse kan følges gennem hele processen med retinogenesis, som er afsluttet i den centrale nethinden i zebrafisk med 3 d postfertilization ( dpf) 4, 5,ref &quot;&gt; 6, 7. Desuden kan de funktionelle krav i forskellige gener i hver af de ovennævnte faser skal samtidig vurderes i zebrafisk nethinden, hvilket giver en fordel frem for andre hvirveldyr modeller, hvor fænotyper som følge af anvendelsen af ​​gen-knockout teknikker kan kun vurderes ved obduktion af fast væv. Navnlig anvendelsen af ​​transgene linjer hvor vi kan visualisere og overvåge ekspressionen af ​​fluorescerende proteiner som reporter transgener i nethinden, giver os mulighed for at opnå tidsmæssige opløsning af genekspression, der ligger til grund for tilblivelsen af ​​en bestemt neuronal celletype. På grund af den hurtige udvikling af zebrafisk, kan disse begivenheder visualiseres under hele udviklingsperiode, hvorved der tilvejebringes dybere indsigt i den tidsmæssige betydning af genekspression i forhold til neuronal erhvervelse celleidentitet og celle adfærd. Endelig kan disse tilgange kombineres effektivt i zebrafisk med frembringelsen af ​​kimære via transplantationer, hvilket resulterer i indsigter i to centrale aspekter af genfunktion. For det første undersøger celler transplanteret fra en donor embryo, hvor et bestemt gen blev slået ned, mens de udvikler i en umærket vildtype miljø, giver os mulighed for at indhente relevante oplysninger om gen-funktion i en celle-uafhængig måde. Dette fører til vigtige indsigter om funktionen af nethinden skæbne afgørende faktorer inden for stamceller de normalt udtrykkes i. Dette eksemplificeres ved undersøgelsen af udviklingsmæssige skæbne resultatet af stamfædre, der ikke længere kan generere funktionelt protein fra disse gener 4, 6, 8, 9. Ved hjælp denne tilgang, har vi vist, at mange skæbne afgørende faktorer (f.eks Vsx1, Atoh7, Ptf1a, Barhl2) handle celle-autonomously at køre specifikke retinale neuronale skæbner; manglen på genekspression fører primært til en skæbne switch, således at cellerne med gen knockdown forbliver levedygtige ved at vedtage en alternativ celleskæbnen 4, 6, 7, 10. For det andet kan sådanne kimære eksperimenter bruges til at vurdere, hvordan vildtype stamfædre opfører sig, når de udvikler inden for forskellige genetiske miljøer. For eksempel ved at sammenligne udviklingen af WT-celler, der normalt udtrykker et gen af interesse (og reporter transgen) i et WT versus manipuleret vært miljø (f.eks gen knockout / knockdown), de resulterende virkninger på genekspression og celleskæbne kan vurderes . Manglen på visse neuron typer i værtsmiljøet, for eksempel har vist sig at påvirke vildtype progenitor adfærd i en celle non-autonomt at forspænde dem til at differentiere til det underrepræsenterede or mangler neuron typer 4, 7, 11, 12. I betragtning af at retinale neuroner er født i en bevaret histogenic ordre ved tidsstyret sekventiel ekspression af specifikke neuronale skæbne determinant gener (skæbne genekspression) (revideret i 13), brugte vi disse metoder til at vise, hvordan timingen af skæbnen genekspression i vildtype stamfædre påvirkes, når disse stamfædre udvikle sig i retinale vært miljøer med inducerede afvigende cellulære kompositioner. Her vil vi skitsere disse tilgange som bevis for, hvordan kombinationen af relativt standard og udbredte teknikker muliggør undersøgelse af timingen af skæbnen genekspression i udviklingen retinale stamfædre 8, 9. Denne protokol beskriver en eksperimenterende tilgang, der kombinerer time-lapse imaging med lethed af prdanner transplantation i ex vivo udvikle zebrafisk embryo at følge individuelle mosaically mærkede celler i hele perioden af udviklingsmæssige retinogenesis. Ved at udføre funktionelle gen manipulationer enten i værten embryo, donor embryo, begge eller ingen af ​​delene, kan man vurdere celle autonomi genfunktion. Denne fremgangsmåde kan tilpasses bredt til lignende forskningsspørgsmål i et andet system, for hvilken de enkelte komponenter, der er skitseret her, er egnede.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Bioline</td>
			<td>BIO-41025</td>
			<td>Agarose coated dishes can be prepared a few days prior, sealed with parafilm to prevent drying and stored at 4C</td>
		</tr>
		<tr>
			<td>Agarose low melt</td>
			<td>Sigma</td>
			<td>A9414-100G</td>
			<td>Can be prepared in larger volume, microwaved to liquify and then kept molte in 40C waterbath. 1 ml aliquots can be prepared (one for each group of embryos mounted).</td>
		</tr>
		<tr>
			<td>borosillicate glass capillary tube w/o filament</td>
			<td>SDR Scientfic</td>
			<td>30-0035</td>
			<td>alternatives with similar diameter can be used&#160;</td>
		</tr>
		<tr>
			<td>borosillicate glass capillary tube with filament</td>
			<td>SDR Scientfic</td>
			<td>30-0038</td>
			<td>alternatives with similar diameter can be used&#160;</td>
		</tr>
		<tr>
			<td>glass petri dishes (60 mm diameter)</td>
			<td>Science Supply</td>
			<td>1070506</td>
			<td>any glass alternative of any size</td>
		</tr>
		<tr>
			<td>Injection tube (2m)</td>
			<td>Eppendorf</td>
			<td>524616004</td>
			<td>This connects the needle holder to the syringe during transplantation</td>
		</tr>
		<tr>
			<td>Microinjection mold (plastic mold with wedge-shaped protrusions)</td>
			<td>Adaptive Science Tools</td>
			<td>PT-1</td>
			<td>alternatives with similar shape can be use</td>
		</tr>
		<tr>
			<td>microneedle holder</td>
			<td>Narishige</td>
			<td>M-152</td>
			<td>any alternative that fits the outside diameter of the injection needles can be used</td>
		</tr>
		<tr>
			<td>microinjector</td>
			<td>Narishige or Eppendorf Femtojet</td>
			<td></td>
			<td>Pneumatic injector using gas pressure</td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Coherent Scientific</td>
			<td>M330IR</td>
			<td>similar alternatives can be used</td>
		</tr>
		<tr>
			<td>microloader tip</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma</td>
			<td>M5904-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>needle puller</td>
			<td>Sutter Instruments</td>
			<td>Model P-2000</td>
			<td>Settings used for this puller are: H 430, Fil 4, Vel 50, Del 225, Pul 75. Any needle puller can be used if it results in appropriate tip dimension</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Interpath</td>
			<td>PM996</td>
			<td>any paraffin film</td>
		</tr>
		<tr>
			<td>Pasteur pipette plastic</td>
			<td>Samco Scientific</td>
			<td>202</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette glass</td>
			<td>Hirschmann Laborgeraete</td>
			<td>9260101</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>Sigma</td>
			<td>P5942-25MG</td>
			<td>&#160;Protease Type XIV</td>
		</tr>
		<tr>
			<td>N-phenyl thiourea</td>
			<td>Sigma</td>
			<td>P7629-25G</td>
			<td>PTU is toxic and can be prepared as a stock solution to avoid frequent exposure to the powder, consult MSDS before purchase / use.</td>
		</tr>
		<tr>
			<td>Qiaquick gel extraction</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td>any alternatives to purify DNA can be used</td>
		</tr>
		<tr>
			<td>Rneasy Mini kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td>any alternatives to purify RNA can be used</td>
		</tr>
		<tr>
			<td>SP6 mMessage kit</td>
			<td>Qiagen</td>
			<td>AM1340</td>
			<td>any alternatives to transcribe DNA using SP6</td>
		</tr>
	</tbody>
</table>

in vivo imaging of transgenic gene expression in individual retinal progenitors in chimeric zebrafish embryos to study cell nonautonomous influences

De genetiske og tekniske styrker har gjort zebrafisk hvirveldyr en central model organisme, hvori konsekvenserne af gen-manipulationer kan spores in vivo hele hurtig udviklingsperiode. Flere processer kan studeres herunder celleproliferation, genekspression, cellemigrering og morfogenese. Vigtigt er det, kan frembringelsen af ​​kimærer gennem transplantationer let udføres, så mosaik mærkning og sporing af individuelle celler under indflydelse af værtsmiljøet. For eksempel kan ved at kombinere funktionelle gen manipulationer af værten embryo (f.eks gennem morpholino mikroinjektion) og levende billedbehandling, virkningerne af ydre, celle nonautonomous signaler (leveret af genetisk modificerede miljø) på individuelle transplanterede donorceller vurderes. Her demonstrerer vi, hvordan denne tilgang anvendes til at sammenligne indtræden af ​​fluorescerende transgen udtryk som en proxy for timingen af ​​cellens skæbne determination i forskellige genetiske værtsmiljøer. I denne artikel giver vi protokollen for mikroinjektion zebrafisk embryoner til at markere donorceller og forårsage gen knockdown i værts embryoner, en beskrivelse af transplantation teknik, der anvendes til at generere kimære embryoner, og protokollen for at forberede og kører i vivo time-lapse konfokal billeddannelse af flere embryoer. Især udfører Multiposition billeddannelse er afgørende, når man sammenligner timingen af ​​begivenheder som indtræden af ​​genekspression. Dette kræver indsamling af data fra flere kontrol og eksperimentelle embryoner behandles samtidigt. En sådan tilgang kan let udvides til studier af ydre påvirkninger i ethvert organ eller væv af valg tilgængelige til at leve billedbehandling, forudsat at transplantationer kan målrettes let i henhold til etablerede embryonale skæbne kort.

Dette arbejde præsenterer en forsøgsprotokol at vurdere ændringer i genekspression timing, når vildtype retinale stamfædre udvikle sig inden en morphant vært foster. De eksperimentelle vært embryoner er Ptf1a morphants, som mangler de mellemliggende født horisontale og amacrine interneuroner i nethinden 7, 26. Disse er blevet sammenlignet med kontrol værten embryoner, som blev injiceret med en standard kontrol morpholino...

Watch this Scientific Journal Video about In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences at JoVE.c

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live-sporing af individuelle WT retinale stamfædre i distinkte genetiske baggrunde giver mulighed for vurdering af bidraget fra celle non-autonome signalering under neurogenese. Her blev en kombination af gen knockdown, kimære generation via embryo transplantation og in vivo time-lapse konfokal billeddannelse anvendes til dette formål.

In Vivo Imaging af transgene genekspression i Individuel Retinal progenitorer i kimære zebrafisk embryoner til Studieinformationen Cell Nonautonomous påvirkninger

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Research

JoVE Journal

Developmental Biology

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

Sporing Celler i GFP-transgene zebrafisk Brug af Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

Kvantitativ analyse af Protein Expression til Studieinformationen Lineage Specification i Mouse præimplantationsperioden Embryoner

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Analyserer<em&gt; In vivo</em&gt; Cell Migration hjælp Cell transplantationer og Time-lapse Imaging i zebrafisk embryoner

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

Instrueret differentiering af Primitive og endelige hæmatopoietisk ophav fra humane pluripotente stamceller

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

Biosensing Motor Neuron Membran Potential 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 ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

Culture of Adult transgene zebrafisk Retinal Eksplantater til Live-celle Imaging af multifoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Anvendelse af RNAi og Heat-shock-induceret transskription faktor udtryk til at omprogrammere kønsceller til neuroner i C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Generation af stillads-fri, tre-dimensionelle Insulin udtrykker Pancreatoids fra mus i bugspytkirtlen stamfaderen In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

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

Visualisere Node og Notochordal plade i Gastrulating mus embryoner ved hjælp af Scanning elektronmikroskopi og hele Mount immunfluorescens

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