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

この作業の一部は、O.S.A.とNSF/BIO助成金1645331 J.L.Rに向けられたUCSDスタートアップファンドによって支えられた。

1. ナッソニア飼育
<ol><li>綿を差し込んだ小さなガラス試験管に、〜20人の合致女性を単独で設置します。 メモ:リアリングスペースを減らすには、10 x 85 cm、4 mLチューブが最適です。女性は、翼が大きく、オビポジターが存在するため、男性と区別されやすい(図1A)。N. ビトリペンシスを飼育するための詳細なプロトコルは、他の文献13、14、15<sup class="xr...

<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>

最近、さまざまな生物学的な質問2、3、7、17のモデル生物としてのナッソニア・ビトリペンシスの使用が増加したことで、N.ビトリペンシス遺伝子の機能解析のための単純化された効率的なプロトコルを可能にする注入方法を開発し、最適化する必要があります。遺伝子編集試薬の胚...

CRISPR/Cas9遺伝子編集は、特にジュエルスズメバチ、ナソニアビトリペンシスなどの多くの上昇モデル生物において、機能的な遺伝学的研究のための強力な技術です。完全なゲノムの入手性と飼育の容易性により、さまざまな生物学的プロセスの分子機構を解明する重要な実験システムとして、宝石のスズイを作ります。例えば、N.ビトリペンシスは最近、ハプロディプロイド性決定系1、2、B染色体3、4、5、6の生物学、概日および季節的な調節7、8の遺伝的基盤のエピジェネティックな基礎を解明するために使用されている。N.ビトリペンニスを使いこなせる機能には、短い世代の時間(25°Cで約2週間)、再生率が高く、パパルステージでの容易な性分離、および4°Cで緊張をジアポーズして保存する機能などがあります。 ライフサイクルは、ブロウフライ、サルコパガ・ブッラタの子犬を寄生する雌のスズメバチから始まります。彼らのオビポジターを介して、メスはブローフライのプパルケースに最大50個の卵を産む。卵は、S.ブラタの子犬を食べる幼虫に発達し、次の数日間にわたって発達し続け、次いで、宿主の子犬9からの成人の飛散および出現を続ける。
N.ビトリペンシスにおいて機能遺伝学的研究を行う分子ツールは、RNA干渉(RNAi)10およびCRISPR/Cas9 11、12など、利用可能であるが、限定的であるが、主に胚性マイクロインジェクション13を行う上で困難なためである。N.ビトリペンニスの卵は開発のためにプパルホストを必要とするので、卵操作は非常に困難です。プレブラトデドムステージ胚は、宿主のブローフライの子犬から採取し、迅速にマイクロインジェクションし、すぐに開発13のために宿主に戻さなければならない。これらのステップは、マイクロ注入胚またはプパルホスト13を損傷することを避けるために、精度と専門的な訓練を必要とします。また、卵は非常に小さく、壊れやすい、特にマイクロインジェクションの後、非常に粘性の細胞質が注射針13の連続的な詰まりを引き起こす。これらの機能は、胚性マイクロインジェクションを非常に困難にし、ほとんどのN.ビトリペニス研究所に存在しない高度な訓練を受けたオペレータと特殊な機器を必要とします。
CRISPR試薬の送達のための代替注入方法の最適化は、モデル生物としてのN.ビトリペンシスの統合に寄与するであろう。子犬と大人の操作は、胚を操作するよりも難しくなく、基本的な注射のセットアップで達成することができます。ここでは、2つのプロトコルは、子犬と成人の注射のために記述されている:注射のための特殊な機器を含む1つと、ガラス毛細血管針を装着したアスピレーターチューブアセンブリの使用を含む他。アスピレーターチューブの使用は、胚マイクロインジェクションのための特殊な機器へのアクセスを持たない実験室に特に適しています。白または黒の子犬および成人のスズメバチを含むN.ビトリペンシスの異なる発達段階の効率的な注射が実証されている。白いプパルの段階でのスズメバチはRNAi媒介性のノックダウン実験に特に適している。ナソーニアのRNAiは2006年10年にリンチとデスプランによって最初に説明されましたが、RNAi注射がどのように行われるかについて利用可能な視覚的な手順はありません。RNAiは最近、B染色体PSR(父性比)3のハプロイダイザー遺伝子を発見し、N.ヴィトリペンシス生物学的リズムにおけるクロック遺伝子の関与を研究するために用いた。
黒い子犬および成体のスズメバチは、ReMOT制御(貨物の受容体媒介性卵巣転移)およびBAPC(分岐性両親媒性ペプチドカプセル)プロトコルを使用してCRISPR/Cas9生殖細胞系列遺伝子編集を誘導するために使用することができる。これら2つの卵巣送達方法は、標的遺伝子において生殖細胞系列変異を発生させるナソニアにおいて有効であると最近説明されている、シナバー7、12。ここでは、ナソニアおよび他の寄生虫スズメバチで機能的遺伝学研究を生成するために利用できるパパルおよび成人注射のステップバイステップ方法論の視覚的手順を含む注射のための単純化されたプロトコルが提供され、特殊な機器を必要とせず、胚微小注射をバイパスする。

<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

ジュエルスズメ バチ、ナソーニアビトリペンニスは、ハプロ-ジプロイド性決定、B染色体生物学、宿主共生相互作用、種分化、および毒合成のエピジェネティクスを研究するための効率的なモデルシステムとなっています。CRISPR/Cas9を含むいくつかの分子ツールが利用可能であるにもかかわらず、機能的遺伝学的研究はこの生物において依然として限られている。 N.ビトリペニス にCRISPR/Cas9技術を適用する主な制限は、胚性マイクロインジェクションの課題に起因する。胚の注射は、この生物では特に困難であり、一般的には多くの寄生虫スズメバチでは、小さな胚の大きさと胚発生のための宿主の子犬の要件のために困難である。これらの課題に対処するために、胚性マイクロインジェクションではなく、成人注射による女性卵巣へのCas9リボヌクレオプロテイン複合体の送達が最適化され、体細胞性および遺伝性生殖細胞系列の両方の編集が得られた。注射手順は、ReMOTコントロール(受容体媒介性貨物の卵巣伝達)またはBAPC(分岐性両親媒性ペプチドカプセル)のいずれかを使用して、子犬および雌のスズメバチで最適化されました。これらの方法は、胚注射に有効な代替手段であることが示されており、部位特異的および遺伝性生殖細胞系列突然変異を可能にする。

この論文では、フェムトジェットまたはアスピレーターチューブを使用して、プパルと成人マイクロインジェクションの2つの簡単な方法を紹介します。第1の方法は、RNAiの一貫性のために重要である液体のより正確な注入を可能にするが、2番目の方法は 、ナソニア の子犬または成人に大量の液体を注入することを可能にする。
<p class="jove_content" fo:keep-togeth...

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

ここでは、 ナソニアビトリペンシス での効率的なプパルおよび成人注射を胚微小注射の代替手段として説明し、RNA干渉(RNAi)またはCRISPR/Cas9ゲノム編集によるRNAサイレンシングまたは遺伝子ノックアウトのいずれかを使用して目的の遺伝子の機能解析を可能にする。

ナソニアビトリペニスにおけるRNAiおよびCRISPR遺伝子編集のためのプパルおよび成人注射

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

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

成虫（A. gambiae）におけるRNAiのアッセイのためのプロトコル

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

生物学

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

ローカライズされたRNAiと創薬リーチにおける外来遺伝子の発現が

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

のパラフィン包埋して凍結切片ショウジョウバエアダルトの筋肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

RNAiは、Necromenic虫にマイクロインジェクションによる遺伝子ノックダウンとトランスジェネシスを媒介 Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

RNAiによる遺伝子ノックダウンと<emインビボで&gt;</em大人の女性の&gt;利尿アッセイ<em&gt;ネッタイシマカ</em&gt;蚊

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

マウスにおける急速な非侵襲腰椎髄腔内注射を経由して生体内 siRNAトランスフェクションと脊髄における遺伝子ノックダウン

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

Intravenous Injections in Neonatal Mice

新生児マウスにおける静脈注射

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

火花のサイレンシング:弱い電気魚のCRISPR/Cas9ゲノム編集

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

単核RNAシーケンシングのための成体マウス腎臓からの核分離

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

ショウジョウバエの蛹の解剖学的、固定、視覚化

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent ...