Name: pupal and adult injections for rnai and crispr gene editing in nasonia vitripennis
Uploaded: 2020-12-04T14:00:00
Description: Watch this Scientific Journal Video about Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis at JoVE.com

Dette arbeidet ble delvis støttet av UCSDs oppstartsfond rettet mot O.S.A. og NSF/BIO grant 1645331 til J.L.R.

1. Nasonia oppdrett
<ol><li>Sett opp ~ 20 parrede kvinner entall i små glassrør plugget med bomull. MERK: For å redusere oppdrettsplassen er 10 x 85 cm, 4 ml rør optimale. Hunnene skiller seg lett fra menn på grunn av de større vingene og tilstedeværelsen av ovipositoren (Figur 1A). Detaljerte protokoller for oppdrett av N. vitripennis finnes også i annen litteratur13,14,15.<ol...

<ol>
	<li>Verhulst, E. C., Beukeboom, L. W., van de Zande, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+control+of+haplodiploid+sex+determination+in+the+wasp+Nasonia.">Maternal control of haplodiploid sex determination in the wasp Nasonia.</a> Science. 328 (5978), 620-623 (2010).</li><li>Verhulst, E. C., Lynch, J. A., Bopp, D., Beukeboom, L. W., van de Zande, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+component+of+the+Nasonia+sex+determining+cascade+is+maternally+silenced+and+regulates+transformer+expression.">A new component of the Nasonia sex determining cascade is maternally silenced and regulates transformer expression.</a> PloS One. 8 (5), 63618 (2013).</li><li>Dalla Benetta, 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=Genome+elimination+mediated+by+gene+expression+from+a+selfish+chromosome.">Genome elimination mediated by gene expression from a selfish chromosome.</a> Science Advances. 6 (14), (2020).</li><li>Aldrich, J. C., Leibholz, A., Cheema, M. S., Ausi&#972;, J., Ferree, P. 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+"selfish"+B+chromosome+induces+genome+elimination+by+disrupting+the+histone+code+in+the+jewel+wasp+Nasonia+vitripennis.">A "selfish" B chromosome induces genome elimination by disrupting the histone code in the jewel wasp Nasonia vitripennis.</a> Scientific Reports. 7, 42551 (2017).</li><li>Ferree, P. 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=Identification+of+genes+uniquely+expressed+in+the+germ-line+tissues+of+the+jewel+wasp+Nasonnia+vitripennis.">Identification of genes uniquely expressed in the germ-line tissues of the jewel wasp Nasonnia vitripennis.</a> G3. 3 (12), 2647-2653 (2015).</li><li>Akbari, O. S., Antoshechkin, I., Hay, B. A., Ferree, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+profiling+of+Nasonia+vitripennis+testis+reveals+novel+transcripts+expressed+from+the+selfish+B+chromosome,+paternal+sex+ratio.">Transcriptome profiling of Nasonia vitripennis testis reveals novel transcripts expressed from the selfish B chromosome, paternal sex ratio.</a> G3. 3 (9), 1597-1605 (2013).</li><li>Dalla Benetta, E., Beukeboom, L. W., van de Zande, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+differences+in+circadian+clock+gene+expression+patterns+and+photoperiodic+diapause+induction+in+Nasonia+vitripennis.">Adaptive differences in circadian clock gene expression patterns and photoperiodic diapause induction in Nasonia vitripennis.</a> The American Naturalist. 193 (6), 881-896 (2019).</li><li>Paolucci, 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=Latitudinal+variation+in+circadian+rhythmicity+in+Nasonia+vitripennis.">Latitudinal variation in circadian rhythmicity in Nasonia vitripennis.</a> Behavioral Sciences. 9 (11), (2019).</li><li>Werren, J. H., Loehlin, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+parasitoid+wasp+Nasonia:+an+emerging+model+system+with+haploid+male+genetics.">The parasitoid wasp Nasonia: an emerging model system with haploid male genetics.</a> Cold Spring Harbor Protocols. 2009 (10), (2009).</li><li>Lynch, J. A., Desplan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+parental+RNA+interference+in+the+wasp+Nasonia+vitripennis.">A method for parental RNA interference in the wasp Nasonia vitripennis.</a> Nature Protocols. 1 (1), 486-494 (2006).</li><li>Li, 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=Generation+of+heritable+germline+mutations+in+the+jewel+wasp+Nasonia+vitripennis+using+CRISPR/Cas9.">Generation of heritable germline mutations in the jewel wasp Nasonia vitripennis using CRISPR/Cas9.</a> Scientific Reports. 7 (1), 901 (2017).</li><li>Chaverra-Rodriguez, D., 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=Germline+mutagenesis+of+Nasonia+vitripennis+through+ovarian+delivery+of+CRISPR-Cas9+ribonucleoprotein.">Germline mutagenesis of Nasonia vitripennis through ovarian delivery of CRISPR-Cas9 ribonucleoprotein.</a> Insect Molecular Biology. , (2020).</li><li>Li, M., Bui, M., Akbari, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo+microinjection+and+transplantation+technique+for+Nasonia+vitripennis+genome+manipulation.">Embryo microinjection and transplantation technique for Nasonia vitripennis genome manipulation.</a> Journal of Visualized Experiments. (130), (2017).</li><li>Werren, J. H., Loehlin, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rearing+Sarcophaga+bullata+fly+hosts+for+Nasonia+(Parasitoid+Wasp).">Rearing Sarcophaga bullata fly hosts for Nasonia (Parasitoid Wasp).</a> Cold Spring Harbor Protocols. 2009 (10), (2009).</li><li>Werren, J. H., Loehlin, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain+maintenance+of+Nasonia+vitripennis+(Parasitoid+Wasp).">Strain maintenance of Nasonia vitripennis (Parasitoid Wasp).</a> Cold Spring Harbor Protocols. 2009 (10), (2009).</li><li>Beukeboom, L. W., van de Zande, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+sex+determination+in+the+haplodiploid+wasp+Nasonia+vitripennis+(Hymenoptera:+Chalcidoidea).">Genetics of sex determination in the haplodiploid wasp Nasonia vitripennis (Hymenoptera: Chalcidoidea).</a> Journal of Genetics. 89 (3), 333-339 (2010).</li><li>Leung, K., van de Zande, L., Beukeboom, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life-history+traits+of+the+Whiting+polyploid+line+of+the+parasitoid.">Life-history traits of the Whiting polyploid line of the parasitoid.</a> Entomologia experimentalis et applicata. 167 (7), 655-669 (2019).</li><li>Kennerdell, J. R., Carthew, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+dsRNA-mediated+genetic+interference+to+demonstrate+that+frizzled+and+frizzled+2+act+in+the+wingless+pathway.">Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway.</a> Cell. 95 (7), 1017-1026 (1998).</li><li>Yang, J., Zhao-jun, H. A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficiency+of+different+methods+for+dsrna+delivery+in+cotton+bollworm+(Helicoverpa+armigera).">Efficiency of different methods for dsrna delivery in cotton bollworm (Helicoverpa armigera).</a> Journal of Integrative Agriculture. 13 (1), 115-123 (2014).</li><li>Brown, S., Holtzman, S., Kaufman, T., Denell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+Tribolium+Deformed+ortholog+and+its+ability+to+directly+regulate+Deformed+target+genes+in+the+rescue+of+a+Drosophila+Deformed+null+mutant.">Characterization of the Tribolium Deformed ortholog and its ability to directly regulate Deformed target genes in the rescue of a Drosophila Deformed null mutant.</a> Development Genes and Evolution. 209 (7), 389-398 (1999).</li><li>Hughes, C. L., 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=RNAi+analysis+of+Deformed,+proboscipedia+and+Sex+combs+reduced+in+the+milkweed+bug+Oncopeltus+fasciatus:+novel+roles+for+Hox+genes+in+the+hemipteran+head.">RNAi analysis of Deformed, proboscipedia and Sex combs reduced in the milkweed bug Oncopeltus fasciatus: novel roles for Hox genes in the hemipteran head.</a> Development. 127 (17), 3683-3694 (2000).</li><li>Dzitoyeva, S., Dimitrijevic, N., Manev, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-abdominal+injection+of+double-stranded+RNA+into+anesthetized+adult+Drosophila+triggers+RNA+interference+in+the+central+nervous+system.">Intra-abdominal injection of double-stranded RNA into anesthetized adult Drosophila triggers RNA interference in the central nervous system.</a> Molecular Psychiatry. 6 (6), 665-670 (2001).</li><li>Chaverra-Rodriguez, D., 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=Targeted+delivery+of+CRISPR-Cas9+ribonucleoprotein+into+arthropod+ovaries+for+heritable+germline+gene+editing.">Targeted delivery of CRISPR-Cas9 ribonucleoprotein into arthropod ovaries for heritable germline gene editing.</a> Nature Communications. 9 (1), 3008 (2018).</li><li>Macias, V. 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=Cas9-mediated+gene-editing+in+the+malaria+mosquito+anopheles+stephensi+by+ReMOT+control.">Cas9-mediated gene-editing in the malaria mosquito anopheles stephensi by ReMOT control.</a> G3. 10 (4), 1353-1360 (2020).</li><li>Heu, C. C., McCullough, F. M., Luan, J., Rasgon, J. L. <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+genome+editing+in+the+silverleaf+whitefly+(Bemisia+tabaci).">CRISPR/Cas9-based genome editing in the silverleaf whitefly (Bemisia tabaci).</a> CRISPR Journal. 3 (2), 89-96 (2020).</li><li>Shirai, Y., Daimon, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+cardinal+are+responsible+for+the+red-1+and+peach+eye+color+mutants+of+the+red+flour+beetle+Tribolium+castaneum.">Mutations in cardinal are responsible for the red-1 and peach eye color mutants of the red flour beetle Tribolium castaneum.</a> Biochemical and Biophysical Research Communications. 529 (2), 372-378 (2020).</li><li>Hunter, W. B., Gonzalez, M. T., Tomich, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAPC-assisted+CRISPR/Cas9+system:+targeted+delivery+into+adult+ovaries+for+heritable+germline+gene+editing+(Arthropoda:+Hemiptera).">BAPC-assisted CRISPR/Cas9 system: targeted delivery into adult ovaries for heritable germline gene editing (Arthropoda: Hemiptera).</a> bioRxiv. , 478743 (2018).</li><li>Chaverra Rodriguez, D., Bui, M., Li, M., Raban, R., Akbari, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+genetic+tools+to+control+ACP.">Developing genetic tools to control ACP.</a> Citrus Research Board Citrograph Magazine. 11 (1), 64-68 (2020).</li></ol>

Med den nylig økte bruken av Nasonia vitripennis som modellorganisme for ulike biologiske spørsmål2,3,7,17, er det behov for å utvikle og optimalisere injeksjonsmetoder for å muliggjøre en forenklet og effektiv protokoll for funksjonell analyse av N. vitripennis gener. De nåværende metodene som involverer embryonal mikroinjeksjon av genredigeringsreagenser er utfordren...

CRISPR/Cas9 genredigering er en kraftig teknologi for funksjonelle genetiske studier, spesielt i mange stigende modellorganismer som juvel veps, Nasonia vitripennis. Den enkle oppdretten og tilgjengeligheten av et komplett genom gjør juvelvevepsen til et viktig eksperimentelt system for å belyse molekylære mekanismer i forskjellige biologiske prosesser. For eksempel har N. vitripennis nylig blitt brukt til å avdekke det epigenetiske grunnlaget for haplodiploid sexbestemmelsessystem1,2, biologien til B-kromosomene3,4,5,6og det genetiske grunnlaget for circadian og sesongregulering7,8. Noen av funksjonene som gjør N. vitripennis egnet til å jobbe med inkluderer kort generasjons tid (~ 2 uker ved 25 °C), høye reproduksjonshastigheter, enkel kjønnsseparasjon på puppetrinnet og evnen til å diapause og lagre stammer ved 4 °C. Livssyklusen begynner med kvinnelige hvepe som parasiterer blowflyens pupper, Sarcophaga bullata. Gjennom sin ovipositor legger kvinner opptil 50 egg i puppetuiet av blowflyen. Egg utvikler seg til larver som spiser på S. bullata pupa, fortsetter å utvikle seg i løpet av de neste dagene, og deretter puperer, etterfulgt av voksen eklosjon og fremvekst fra verten puparium9.
Molekylære verktøy for å utføre funksjonelle genetiske studier i N. vitripennis, for eksempel RNA-interferens (RNAi)10 og CRISPR / Cas911,12, er tilgjengelige, men er begrenset, hovedsakelig på grunn av vanskeligheter med å utføre embryonale mikroinjeksjoner13. Siden N. vitripennis egg krever en pupal vert for utvikling, egg manipulasjon er svært utfordrende. Pre-blastoderm stadium embryoer må samles fra verten blowfly pupper, raskt mikroinjisert, og umiddelbart overført tilbake til verten for utvikling13. Disse trinnene krever presisjon og spesialisert trening for å unngå å skade de mikroinjiserte embryoene eller puppevertene13. Videre er eggene svært små og skjøre, spesielt etter mikroinjeksjoner, med en veldig viskøs cytoplasma som forårsaker en kontinuerlig tilstopping av injeksjonsnålen13. Disse funksjonene gjør embryonale mikroinjeksjoner usedvanlig utfordrende, og krever høyt utdannede operatører og spesialisert utstyr som er fraværende i de fleste N. vitripennis laboratorier.
Optimalisering av alternative injeksjonsmetoder for levering av CRISPR-reagenser vil bidra til konsolidering av N. vitripennis som modellorganisme. Manipulering av pupper og voksne er mindre utfordrende enn å manipulere embryoer og kan oppnås med et grunnleggende injeksjonsoppsett. Her er to protokoller beskrevet for injeksjon av pupper og voksne: den ene involverer spesialisert utstyr for injeksjoner, og den andre involverer bruk av en aspiratorrørmontering utstyrt med en glasskapillær nål. Bruken av et aspiratorrør er spesielt egnet for laboratorier som ikke har tilgang til spesialisert utstyr for embryomikroinjeksjoner. Effektive injeksjoner av ulike utviklingsstadier av N. vitripennis, inkludert hvit eller svart pupper og voksenveps, demonstreres. Wasps på den hvite puppetrinnet er spesielt egnet for RNAi-medierte knockdown-eksperimenter. Selv om RNAi i Nasonia først ble beskrevet av Lynch og Desplan i 200610, er det ingen visuell prosedyre tilgjengelig for hvordan RNAi-injeksjoner utføres. RNAi ble nylig brukt til å oppdage haploidizer genet av B-kromosomet PSR (Paternal Sex Ratio)3 og for å studere involvering av klokkegenet, periode, i N. vitripennis biologiske rytmer7.
Svart pupper og voksne veps kan brukes til å indusere CRISPR/Cas9 germline genredigering ved hjelp av ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) og BAPC (Branched Amphiphilic Peptide Capsules) protokoller. Disse to eggstokkleveringsmetodene har nylig blitt beskrevet å være effektive i Nasonia for å generere bakteriemutasjoner i målgenet, cinnabar7,12. Her er en forenklet protokoll gitt for injeksjoner, inkludert en visuell prosedyre for en trinnvis metodikk for både pupal og voksen injeksjoner som kan brukes til å generere funksjonelle genetikkstudier i Nasonia og sannsynligvis i andre parasitoidveps, uten å kreve spesialisert utstyr og omgå embryonale mikroinjeksjoner.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 x 15 mm Stackable Petri Dishes, Polystyrene, Mono, Sterile</td>
			<td>Sigma</td>
			<td>960-97693-083</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminosilicate glass capillary tubing 1 mm(outside diameter) x 0.58 mm (inner diameter)</td>
			<td>Sutter Instruments</td>
			<td>BF100-58-10</td>
			<td>Can also use Borosilicate or Quartz</td>
		</tr>
		<tr>
			<td>Aspirator tube assemblies for calibrated microcapillary pipettes</td>
			<td>Sigma</td>
			<td>A5177-5EA</td>
			<td></td>
		</tr>
		<tr>
			<td>BAPC</td>
			<td>phoreus biotech</td>
			<td>BAPtofect-25 0.5 mg Kit</td>
			<td></td>
		</tr>
		<tr>
			<td>Cas9 Protein with NLS</td>
			<td>PNABio</td>
			<td>CP01</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting needle</td>
			<td>VWR</td>
			<td>10806-330</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase/RNase-Free distilled Water</td>
			<td>Invitrogen</td>
			<td>10977-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Femtojet Express programmable microinjector</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Femtotips Microloader tips</td>
			<td>Fisher Scientific</td>
			<td>E5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine-tip paintbrush</td>
			<td>ZEM</td>
			<td>2595</td>
			<td></td>
		</tr>
		<tr>
			<td>Flesh fly pupae,&#160;Sarcophaga bullata</td>
			<td>Ward&#39;s Science</td>
			<td>470180-392</td>
			<td></td>
		</tr>
		<tr>
			<td>Food colorant dye</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Test Tubes</td>
			<td>Fisher Scientific</td>
			<td>982010</td>
			<td></td>
		</tr>
		<tr>
			<td>Glue</td>
			<td>Elmer&#39;s</td>
			<td></td>
			<td>washable, no toxic school glue</td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>P-1000 or P-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>Fisherbrand</td>
			<td>12-550-A3</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>SZ51</td>
			<td></td>
		</tr>
	</tbody>
</table>

pupal and adult injections for rnai and crispr gene editing in nasonia vitripennis

Juvelvevepsen, Nasonia vitripennis, har blitt et effektivt modellsystem for å studere epigenetikk av haplo-diploid sexbestemmelse, B-kromosombiologi, vertssympymiske interaksjoner, spesiering og giftsyntese. Til tross for tilgjengeligheten av flere molekylære verktøy, inkludert CRISPR / Cas9, er funksjonelle genetiske studier fortsatt begrenset i denne organismen. Den største begrensningen ved bruk av CRISPR/Cas9-teknologi i N. vitripennis stammer fra utfordringene ved embryonale mikroinjeksjoner. Injeksjoner av embryoer er spesielt vanskelig i denne organismen og generelt i mange parasitoidveps, på grunn av liten embryostørrelse og kravet om en vertsvalpe for embryonal utvikling. For å løse disse utfordringene ble Cas9 ribonucleoprotein kompleks levering til kvinnelige eggstokkene ved vokseninjeksjon, i stedet for embryonal mikroinjeksjon, optimalisert, noe som resulterte i både somatiske og arvelige bakterieredigeringer. Injeksjonsprosedyrene ble optimalisert i pupper og kvinnelige veps ved hjelp av enten ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) eller BAPC (Branched Amphiphilic Peptide Capsules). Disse metodene viser seg å være effektive alternativer til embryoinjeksjon, noe som muliggjør stedsspesifikke og arvelige bakteriemutasjoner.

Dette papiret presenterer to enkle metoder for pupal og voksen mikroinjeksjon, enten ved hjelp av en femtojet eller et aspiratorrør. Den første metoden tillater en mer presis injeksjon av væske, noe som er viktig for RNAi-konsistens, mens den andre tillater injeksjon av større mengder væske i Nasonia-pupper eller voksne.
Representative resultater presentert i tabell 1 viser god overlevelsesrate (fr...

Watch this Scientific Journal Video about Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis at JoVE.com

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

Her beskriver vi metoder for effektive pupal- og vokseninjeksjoner i Nasonia vitripennis som tilgjengelige alternativer til embryomikroinjeksjon, noe som muliggjør funksjonell analyse av gener av interesse ved hjelp av enten RNA-silencing via RNA-interferens (RNAi) eller gen knockout via CRISPR / Cas9 genomredigering.

Pupal og voksen injeksjoner for RNAi og CRISPR Genredigering i Nasonia vitripennis

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, ...

Research on non-canonical insect model, like parasitoid wasp, is limited because embryonic microinjection is very challenging. Our method bypasses this problem, enabling site-specific and heritable germline mutations. Injecting pupae or adult females is fast and removes the requirement for expensive equipment or specialized training required to achieve successful embryonic microinjections.This technique will allow no specialized labs that do not have access to expensive equipment to do functional studies in any insect species they are using as a model. After the optimization, this method can be used to study functional genetics of many other genes in Nasonia. We also think this method may be used to deliver nucleic acids for genetic transformation of Nasonia or other insects.Begin by setting up approximately 20 mated females singularly in small glass test tubes plugged with cotton. Add two S.bullata hosts per tube and maintain the wasps at 25 degrees Celsius and 30%relative humidity with a 12:12 light dark cycle for two to three days. After two to three days, gently remove the females using a fine tipped brush to avoid continuous over position and a synchrony in offspring development.Removed females can optionally be re-hosted or stored at five degrees Celsius for up to a month. Maintain the parasitized host at 25 degrees Celsius and 30%relative humidity with a 12:12 light dark cycle for seven days if the desired injection stage requires white pupae, for 13 days, if the required developmental stage is black pupae, or 14 days for young, newly emerged adults. When the desired stage is reached, crack open the host puparium with a dissecting needle to recover N.vitripennis pupae and adults.Prepare a glass slide by applying a line of school glue at the center. Spread the glue with the dissecting needle to obtain a thick layer, which will be used to align the pupae. Let the glue dry for approximately two minutes before transferring the pupae.Under a dissecting microscope, use a dissecting needle to apply glue on the back of the head of the pupa. Attach the pupa to the glue layer on the slide with its abdomen facing up. Align 20 to 30 pupae side-by-side on the slide.Place the slide with the pupae and a Petri dish to let the glue drive for approximately 10 minutes. Before starting the injections, test the adherence of the pupae to the glue by pushing them gently with the dissecting needle. If most of the pupae are loosened, prepare a new slide.Alternatively, remove all loose pupae and proceed to inject those that are properly attached to the slide. Load one capillary glass tube into a needle puller and pull needles following the manufacturer's instructions. Load the needle with five microliters of the prepared injection mixture using microloader pipette tips.Open the needle, sliding the tip on a surface made from two overlapping slides. Alternatively, open the tip of the needle with a pair of fine forceps to create a sharp edge. Place the slide with the aligned pupae under a dissecting microscope, then insert the needle into the femtojet's injection tube and tighten the grip head.Looking at the needle under the microscope, turn on the femtojet and set the compensation pressure and injection pressure parameters by rotating the rotary knobs. Carefully insert the needle between the second and third visible abdominal segments with a vertical angle of about 30 degrees. Inject with a continuous flow until the whole abdomen turns green in the case of white stage pupae or until the abdomen increases in size in the case of black pupae.Stop injecting when it is clear that the abdomen cannot take any more liquid or when the liquid starts flowing out of the body. Move carefully to the next pupa and repeat these steps. Transfer the slide with the injected pupae to a Petri dish.Cover the dish with its lid and place it at 25 degrees Celsius until wasp emergence. For adult preparation, separate groups of 20 virgin females in a clean small test tube with a fine paint brush. Place a glass slide on top of ice and align the female side by side on the cold slide with abdomens facing up under a dissecting scope.Load one needle with the ribonucleoprotein solution using a microloading tip and insert the needle into the connector pack of the aspirator tube assembly. Support the females from the opposite side with blunted dissecting needles, while injecting slowly into the abdomen. Avoid touching the ovipositor.This will severely damage this critical reproductive structure. Carefully insert the needle between the second and third visible abdominal segments with a vertical angle of about 30 degrees. Inject the solution into the female abdomen, stopping when no more liquid can enter or when leaking of surplus solution is observed.Move carefully to the next female and repeat these steps. When finished, gently transfer a single injected females to a new tube with one host using a paintbrush. Leave them to recover at room temperature for approximately one hour, confirm survival, and return the tubes to the rearing incubator.After adult eclosion from the injected pupae, place single wasps into a glass to plugged with cotton and insert one S.bullata host. For CRISPR/Cas9 knockout experiments, allow females to parasitize the host for one day and replace the host each day for three consecutive days. Place the parasitized host, set 25 degrees Celsius until emergence of the G0 male offspring, approximately 13 to 14 days.Under a dissecting microscope, screen G0 males for the mutated phenotypes. The cinnabar gene is responsible for eye pigmentation. Wasps with brown eyes are wild-type and wasps with bright red eyes or variations between red and brown eyes are mutants.This protocol can be used for either pupal or adult microinjection. Survival rates of this procedure range from 15 to 100%The efficiency of achieving the desired phenotype via RNAi or CRISPR without requiring laborious egg injections is shown here. When injecting pupae or adult females, the needle should be sharp and be introduced in a way that liquid should not leak immediately after injecting.If this happens, remove the needle, ensure that the tip is fine and sharp, change it if necessary or inject in a different location. Function analysis can be performed on the gene of interest targeted from this procedure. This will answer a question about the gene function and if the gene is necessary for the specific phenotype studied.

Research

JoVE Journal

Biology

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

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