Name: egg microinjection and efficient mating for genome editing in the firebrat thermobia domestica
Uploaded: 2020-10-20T15:00:00
Description: Watch this Scientific Journal Video about Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica at JoVE.com

TO og TD blev støttet af JSPS KAKENHI tilskud numre 19H02970 og 20H02999, hhv.

1. Vedligeholdelse af laboratoriekolonier
<ol><li>Til vedligeholdelse af vilde og mutantpopulationer skal du bruge en stor plastbeholder (460 mm x 360 mm x 170 mm) med almindelig kunstig fiskefoder, vand i plastikkopper med et ventilationshul på toppen, et foldet papir til at skjule insekterne og lagdelt bomuld til æglægning (Figur 2A). Hold alle T. domestica kulturer inde i 37 °C inkubatorer og sæt den relative luftfugtighed (RH) inde i hver beholder til 60%-80%. BEMÆRK:...

<ol>
	<li>Peterson, M. D., Rogers, B. T., Popadi&#263;, A., Kaufman, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+embryonic+expression+pattern+of+labial,+posterior+homeotic+complex+genes+and+the+teashirt+homologue+in+an+apterygote+insect.">The embryonic expression pattern of labial, posterior homeotic complex genes and the teashirt homologue in an apterygote insect.</a> Development Genes and Evolution. 209, 77-90 (1999).</li><li>Farris, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+organization+of+the+mushroom+bodies+of+Thermobia+domestica+(Zygentoma,+Lepismatidae):+Insights+into+mushroom+body+evolution+from+a+basal+insect.">Developmental organization of the mushroom bodies of Thermobia domestica (Zygentoma, Lepismatidae): Insights into mushroom body evolution from a basal insect.</a> Evolution and Development. 7, 150-159 (2005).</li><li>Kamae, Y., Tanaka, F., Tomioka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+and+functional+analysis+of+the+clock+genes,+Clock+and+cycle,+in+the+firebrat+Thermobia+domestica.">Molecular cloning and functional analysis of the clock genes, Clock and cycle, in the firebrat Thermobia domestica.</a> Journal of Insect Physiology. 56, 1291-1299 (2010).</li><li>Ohde, T., Masumoto, M., Yaginuma, T., Niimi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+RNAi+analysis+in+the+firebrat,+Thermobia+domestica:+Distal-less+is+required+to+form+caudal+filament.">Embryonic RNAi analysis in the firebrat, Thermobia domestica: Distal-less is required to form caudal filament.</a> Journal of Insect Biotechnology and Sericology. 105, 99-105 (2009).</li><li>Ohde, T., Yaginuma, T., Niimi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nymphal+RNAi+analysis+reveals+novel+function+of+scalloped+in+antenna,+cercus+and+caudal+filament+formation+in+the+firebrat,+Thermobia+domestica.">Nymphal RNAi analysis reveals novel function of scalloped in antenna, cercus and caudal filament formation in the firebrat, Thermobia domestica.</a> Journal of Insect Biotechnology and Sericology. 108, 101-108 (2012).</li><li>Bell&#233;s, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+Drosophila:+RNAi+in+vivo+and+functional+genomics+in+insects.">Beyond Drosophila: RNAi in vivo and functional genomics in insects.</a> Annual Review of Entomology. 55, 111-128 (2010).</li><li>Ohde, T., Takehana, Y., Shiotsuki, T., Niimi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-based+heritable+targeted+mutagenesis+in+Thermobia+domestica:+A+genetic+tool+in+an+apterygote+development+model+of+wing+evolution.">CRISPR/Cas9-based heritable targeted mutagenesis in Thermobia domestica: A genetic tool in an apterygote development model of wing evolution.</a> Arthropod Structure and Development. 47, 362-369 (2018).</li><li>Nji, 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=CRISPR/Cas9+in+insects:+Applications,+best+practices+and+biosafety+concerns.">CRISPR/Cas9 in insects: Applications, best practices and biosafety concerns.</a> Journal of Insect Physiology. 98, 245-257 (2017).</li><li>Brand, P., 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=The+origin+of+the+odorant+receptor+gene+family+in+insects.">The origin of the odorant receptor gene family in insects.</a> eLife. 7, 1-13 (2018).</li><li>Noble-Nesbitt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water+balance+in+the+firebrat,+Thermobia+domestica+(Packard).+Exchanges+of+water+with+the+atmosphere.">Water balance in the firebrat, Thermobia domestica (Packard). Exchanges of water with the atmosphere.</a> Journal of Experimental Biology. 50, 745-769 (1969).</li><li>Sweetman, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+ecology+of+the+firebrat,+Thermobia+domestica+(Packard).">Physical ecology of the firebrat, Thermobia domestica (Packard).</a> Ecological Monographs. 8, 285-311 (1938).</li><li>Nijhout, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Insect Hormones. , (1994).</li><li>Chen, 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=Efficient+detection,+quantification+and+enrichment+of+subtle+allelic+alterations.">Efficient detection, quantification and enrichment of subtle allelic alterations.</a> DNA Research. 19, 423-433 (2012).</li><li>Mashal, R. D., Koontz, J., Sklar, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+mutations+by+cleavage+of+DNA+heteroduplexes+with+bacteriophage+resolvases.">Detection of mutations by cleavage of DNA heteroduplexes with bacteriophage resolvases.</a> Nature Genetics. 9, 177-183 (1995).</li><li>Adams, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+notes+upon+the+firebrat,+Thermobia+domestica+Packard.">Biological notes upon the firebrat, Thermobia domestica Packard.</a> Journal of the New York Entomological Society. 41, 557-562 (1933).</li></ol>

For en vellykket generation af den ønskede T. domestica mutant med CRISPR/Cas9 er det først vigtigt at indsamle et tilstrækkeligt antal iscenesatte embryoner til injektion. For en konstant samling af et tilstrækkeligt antal T. domestica æg er nøglen at vælge en passende størrelse af beholderen for at have en lavere befolkningstæthed, fordi det ville hjælpe med en vellykket færdiggørelse af en række komplekse parringsadfærd, som gentages efter hver voksen smeltet13. ...

Thermobia domestica tilhører en af de mest basale insektordrer, Zygentoma, som bevarer en forfædres ametaboløs og vingeløs livscyklus. En sådan basal fylogenetisk position og forfædres egenskaber sætter denne art som en attraktiv model til undersøgelse af de mekanismer, der ligger til grund for insekternes succes på Jorden, som dækker over 70% af de beskrevne dyrearter1. T. domestica har længe hovedsagelig været anvendt til at studere insektfysiologiens forfædres egenskaber på grund af dens egnede egenskaber som laboratoriemodel, såsom en relativt kort livscyklus (2,5-3,0 måneder fra embryo til reproduktiv voksen; Figur 1A) og en nem avl. I de sidste tre årtier er dets anvendelse blevet udvidet til at undersøge forfædres egenskaber ved forskellige træk som kropsplan, neural differentiering og døgnrytme2,3,4.
Anvendelsen af avancerede genetiske værktøjer i T. domestica kunne yderligere fremskynde sådanne bidrag på et bredt forskningsområde. Vellykket RNA interferens (RNAi)-medieret gen knockdown i embryoner, nymfer, og voksne er blevet rapporteret i T. domestica4,5,6. Effektiviteten af systemisk RNAi er stadig meget artsafhængig - for eksempel er den generelt høj i coleoptera, mens den er lav i lepidoptera-rækkefølgen7. Effektiviteten og varigheden af RNAi knockdown i T. domestica er endnu ikke vurderet. Ud over RNAi har vi tidligere rapporteret en vellykket CRISPR/Cas9-medieret gen knockout i T. domestica8. CRISPR/Cas-systemet er i vid udstrækning blevet anvendt til genomredigering i insekter, især til målrettet gen knockout. Dens anvendelse kunne udvides til andre applikationer såsom gen reporter assay, celle afstamning tracking, og manipulation af transskriptionel aktivitet ved at banke-in udefrakommende konstruktioner efter oprettelsen af en protokol for levering af komponenter i CRISPR / Cas-systemet i kerne9. Kombineret med den offentliggjorte genomsamling10vil den brede anvendelse og videreudvikling af den CRISPR/Cas-baserede genomredigering i T. domestica lette undersøgelser med fokus på de evolutionære mekanismer bag insekternes enestående adaptive succes. Her beskriver vi en detaljeret protokol for embryomikroinjection og for parring af voksen T. domestica for at generere en mutant stamme ved hjælp af CRISPR/Cas9. I betragtning af denne nye metode diskuterer vi vigtigheden af at overveje den unikke biologi af ikke-traditionelle modelarter til vellykkede anvendelser af disse teknikker.

<table>
	<tbody>
		<tr>
			<td>24-well plate</td>
			<td>Corning</td>
			<td>83-3738</td>
			<td></td>
		</tr>
		<tr>
			<td>Alt-R S.p. HiFi Cas9 Nuclease V3, 100 &#181;g</td>
			<td>Integrated DNA Technologies</td>
			<td>1081060</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-static cleaner</td>
			<td>Hozan</td>
			<td>Z-292</td>
			<td>for removing static electricity from a 24-well plate</td>
		</tr>
		<tr>
			<td>Barrier Box 20.7L</td>
			<td>AS ONE</td>
			<td>4-5606-01</td>
			<td>Large container</td>
		</tr>
		<tr>
			<td>FemtoJet 4i</td>
			<td>Eppendorf</td>
			<td>5252000013</td>
			<td>Electronic microinjector</td>
		</tr>
		<tr>
			<td>Femtotip II, injection capillary</td>
			<td>Eppendorf</td>
			<td>5242957000</td>
			<td>Glass injection capillary</td>
		</tr>
		<tr>
			<td>High Pack 2440mL</td>
			<td>AS ONE</td>
			<td>5-068-25</td>
			<td>Middle-sized container</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Panasonic</td>
			<td>MIR-554-PJ</td>
			<td>for 37 &#176;C incubation. No need to humidify inside the incubator.</td>
		</tr>
		<tr>
			<td>KOD Fx Neo</td>
			<td>Toyobo</td>
			<td>KFX-101</td>
			<td>PCR enzyme for genotyping. Optimized for an amplification from crude templates.</td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td>Narishige</td>
			<td>GJ-8</td>
			<td>for holding the micromanipulator</td>
		</tr>
		<tr>
			<td>Microloader</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Narishige</td>
			<td>MM-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>SZX12</td>
			<td>for microinjection. More than 35X magnification is sufficient for the microinjection</td>
		</tr>
		<tr>
			<td>MultiNA</td>
			<td>Shimadzu</td>
			<td>MCE-202</td>
			<td>Microchip electrophoresis system</td>
		</tr>
		<tr>
			<td>NiceTac</td>
			<td>Nichiban</td>
			<td>NW-5</td>
			<td>Double-sided tape to place eggs on a glass slide</td>
		</tr>
		<tr>
			<td>Paint brush (horse hair)</td>
			<td>Pentel</td>
			<td>ZBS1-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Plant culture dish</td>
			<td>SPL Life Sciences</td>
			<td>310100</td>
			<td>Mating dish and water supplies for a large and middle-sized containers</td>
		</tr>
		<tr>
			<td>Proteinase K, recombinant, PCR Grade Lyophilizate from Pichia pastoris</td>
			<td>Roche</td>
			<td>3115836001</td>
			<td></td>
		</tr>
		<tr>
			<td>SZX 12 microscope</td>
			<td>Olympus</td>
			<td>SZX 12</td>
			<td>More than 35X magnification is sufficient for the microinjection</td>
		</tr>
		<tr>
			<td>Talcum powder</td>
			<td>Maruishi</td>
			<td>877113</td>
		</tr>
		<tr>
			<td>Tetra Goldfish Gold Growth</td>
			<td>Spectrum Brands</td>
			<td></td>
			<td>Artificial regular fish food</td>
		</tr>
	</tbody>
</table>

egg microinjection and efficient mating for genome editing in the firebrat thermobia domestica

Den firebrat Thermobia domestica er en ametaboløs, vingeløs art, der er egnet til at studere de udviklingsmæssige mekanismer af insekter, der førte til deres vellykkede evolutionære stråling på jorden. Anvendelsen af genetiske værktøjer såsom genomredigering er nøglen til at forstå genetiske ændringer, der er ansvarlige for evolutionære overgange i en Evo-Devo-tilgang. I denne artikel beskriver vi vores nuværende protokol til generering og vedligeholdelse af mutantstammer af T. domestica. Vi rapporterer en tør injektionsmetode som et alternativ til den rapporterede våde injektionsmetode, der giver os mulighed for at opnå stabilt høje overlevelsesrater i injicerede embryoner. Vi rapporterer også en optimeret miljøindstilling for at parre voksne og opnå efterfølgende generationer med høj effektivitet. Vores metode understreger vigtigheden af at tage hensyn til hver arts unikke biologi for en vellykket anvendelse af genomredigeringsmetoder på ikke-traditionelle modelorganismer. Vi forudser, at disse genomredigeringsprotokoller vil hjælpe med at implementere T. domestica som laboratoriemodel og yderligere fremskynde udviklingen og anvendelsen af nyttige genetiske værktøjer i denne art.

I vores hænder kan omkring 100 æg injiceres godt med en enkelt injektion kapillær, når den har tilstrækkelig spids (Figur 3C). Injektion af gRNA/Cas9 ribonucleoproteinkompleks i embryoner inden for de første 8 timer efter æglægning resulterer i indeller på det gRNA-målrettede sted. Dette forårsager biallelic mutationer i nogle celler i den injicerede generation (G0) og dermed mutant mosaik fænotyper er normalt opnået i G0. For eksempel, når denne protokol blev brugt til at inji...

Watch this Scientific Journal Video about Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica at JoVE.com

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

Vi leverer en detaljeret protokol for opdræt, mikroinjection af æg og for effektiv parring af firebrat Thermobia domestica at generere og vedligeholde mutant stammer efter genom redigering.

Egg Microinjection og effektiv parring for Genom Redigering i Firebrat Thermobia domestica

The firebrat Thermobia domestica is an ametabolous, wingless species that is suitable for studying the developmental mechanisms of insects that led to ...

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JoVE Journal

Developmental Biology

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

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Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance ...

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Samling af efter parring sæd fra den kvindelige forplantningsorganerne og målingen af sæd fortætning i mus

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