Name: microinjection of western corn rootworm, diabrotica virgifera virgifera, embryos for germline transformation, or crispr/cas9 genome editing
Uploaded: 2018-04-27T14:30:00
Description: Watch this Scientific Journal Video about Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing at JoVE.com

Dette arbeidet ble støttet av et stipend fra Monsanto korn Rootworm kunnskap Research Program, gi nummer AG/1005 (å MDL og YC) og oppstart midler til MDL fra NCSU. FC ble støttet av tilskudd fra Monsanto korn Rootworm kunnskap Research Program (AG/1005) og National Science Foundation, gi nummer kalt MCB-1244772 (MDL). Forfatterne erklærer ingen konkurrerende interesser. FC og MDL unnfanget og utformet eksperimenter; FC, PW og SP utført eksperimenter; FC og MDL analysert resultatene; og FC, NG og MDL skrev manuskriptet. Vi takker Teresa O'Leary, William Klobasa og Stephanie Gorski for deres eksperthjelp i screening WCR. Vi takker også Dr. Wade French (USDA-ARS, nord sentrale Agricultural Research Laboratory, Brookings, SD) for leveranser av egg å etablere lab kolonier og gir oppdrett protokoller.

1. koloni-nivå oppdrett WCR voksne
<ol><li>Få en tilstrekkelig mengde WCR voksne (500-1000) fra et pålitelig selskap eller forskning laboratorium (se Tabell for materiale for et eksempel) og plasser i en 30 cm3 bur. Merk: Bruk av en ikke-diapausing belastning anbefales sterkt.</li>
	<li>Forberede WCR kunstig kosthold etter produsentens protokoll og hell et 1 cm-tykke lag i en 38 oz beholder (se Tabell for materiale). Etter miksing, lagre ubrukte kosthold på 4 ° C for...

<ol>
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Selv om microinjection av WCR med dsRNAs for å RNAi har vært rapportert9, er den første protokollen å etablere rutiner for microinjecting precellular WCR embryoer, en kritisk prosess for å gjennomføre germline transformasjon og/eller CRISPR/Cas9 genomet redigering i denne arten. Vellykket microinjection av WCR embryo er avhengig av mange faktorer, som er transformasjon effektivitet. Diskuteres nedenfor er noen av de store problemene påvirker utfallet av bruker denne protokollen germline tra...

Vestlige mais rootworm (WCR), Diabrotica virgifera virgifera, er en viktig pest korn1. Interessant, WCR synes å overvinne kontroll måler enn mest landbruket skadedyr siden de ikke bare tilpasse fysiologisk, men også behaviorally2,3,4,5, 6. det siste tiåret, RNA-interferens (RNAi), et kraftig funksjonelle genomisk verktøy har blitt undersøkt som potensielle kontroll WCR7,8, og har også blitt brukt som et middel til å studere genet funksjon9. Men mens RNAi er ofte utført av microinjection av double-strandet RNA (dsRNA) i andre arter, er injeksjon-baserte RNAi sjelden i WCR. Faktisk, er det bare noen rapporter om RNAi via microinjection av dsRNA til WCR9. Grunnen er at WCR kan oppnå høy-nivåer av genet knockdown via inntak av dsRNA7,10, selv tillater studiet av embryonale effekter av mate dsRNA mor11. Mens denne metoden gjør WCR et utmerket emne for genomisk funksjonsanalyse via RNAi, har det bremset fremdriften for utviklingen av metoder for embryonale microinjection i denne arten.
Til tross for kraften i RNAi er det noen ulemper. For eksempel svare ikke alle gener like RNAi. Denne variasjonen kan gjøre tolke resultatene av en funksjonell genomforskning analysen vanskeligere. Også RNAi er forbigående, og genererer ikke arvelig mutasjoner. På den annen side, kan germline transformasjon, en annen funksjonelle genomisk verktøyet, generere arvelige mutasjoner via innsetting av et markert transposable element i genomet12,13. Dette gjør germline transformasjon et utmerket verktøy for å lage mutant stammer for bruk i langsiktige genetiske studier. Transformasjon kan også brukes til å redde en mutasjon ved å levere en funksjonell kopi av genet til en mutant genomet14. Videre er germline transformasjon hjørnesteinen i en rekke molekylær genetisk teknikker. I tillegg til fortrenging og/eller redde gen funksjon, kan germline transformasjon brukes for enhancer overlapping15, gene overlapping16, Gal4-baserte ektopisk uttrykk17og genom hele mutagenese18.
Flere nylig, verktøy sofistikert genomet redigering har blitt utviklet. Disse verktøyene inkluderer transkripsjon Activator-Like effektor Nucleases (TALEN) og CRISPR/Cas9-nuclease systemet19,20. Fordelen med disse nye verktøyene er at de tilbyr forskere evnen til å indusere en double-strandet DNA pause på nesten overalt i nesten alle genom. Når indusert, disse pausene kan repareres via enten ikke-homologe end begynte, som kan presentere slettinger eller innsettinger i målrettede genet, eller homologi-rettet reparasjon, som i nærvær av en konstruert konstruksjonen kan katalysere det utskifting av hele gener eller genetisk regioner21,22. Disse metodene krever imidlertid microinjection DNA, RNA og/eller protein i svært ung embryoer.
Enkelt viktigere, i motsetning til dsRNAs som brukes for RNAi, nucleic syrer og proteiner brukes for germline transformasjon og genom redigering ikke kan krysse cellemembraner. Derfor må microinjection plasmider DNAs, mRNAs og/eller proteiner skje under syncytial blastoderm Stadium (dvs. før insekt embryoet cellularizes). Dette gjør tidspunktet for injeksjoner en kritisk faktor. For eksempel i Drosophila melanogastermå embryo injiseres innen to timer etter egg lå12. Derfor må utviklingen av en vellykket embryonale microinjection protokoll for WCR ta hensyn de beste forholdene for kvinnelige egglegging, og den beste metoden for å samle tilstrekkelig mengder precellular embryoer.
Et forsøk på å bringe en rekke molekylær genetisk verktøy på WCR biologi krever utvikle metoder for innsamling og microinjecting precellular WCR embryoer. Her gir vi detaljerte instruksjoner, tips og triks, å hjelpe andre bruker transformasjon-baserte teknikker i WCR forskning. I tillegg til å utvide transgene teknologier til WCR for bruk i funksjonelle genomisk studier, kan disse teknikkene også kraftige nye pest kontroll strategier som gene stasjonen23,24 skal testes i dette økonomisk viktigste pest.

<table border="0" cellpadding="0" cellspacing="0" width="1155">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Qualitative Filter Paper (Black)</td>
			<td style="width:189px;">Ahlstrom</td>
			<td style="width:287px;">8613-0900</td>
			<td style="width:339px;">For egg microinjection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 oz Containers&#160;</td>
			<td style="width:189px;">Anny&#39;s Plastic Tableware</td>
			<td>ASET101</td>
			<td>Egg container</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drosophila Agar Type II</td>
			<td style="width:189px;">Apex</td>
			<td style="width:287px;">66-103</td>
			<td>Substrate for WCR egg-laying dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Featherweight Forceps&#160;</td>
			<td>Bioquip</td>
			<td style="width:287px;">4748</td>
			<td>Handling larvae and pupae&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clorox Regular-Bleach1</td>
			<td style="width:189px;">Clorox</td>
			<td style="width:287px;">44600-30770</td>
			<td>Wash corn and dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trucker&#39;s Favorite Yellow</td>
			<td>Coor Farm Supply</td>
			<td>502</td>
			<td>Corn for feeding WCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Microinjection System (Homemade)</td>
			<td style="width:189px;">NA</td>
			<td style="width:287px;">Parts &#38; instructions available upon request</td>
			<td>Controls injection pressure (0-20 psi)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Elmer&#39;s Non-toxic Glue</td>
			<td>Elmer&#39;s Products, Inc.</td>
			<td style="width:287px;">E304</td>
			<td>Glue eggs on filter paper</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">epTIPS Microloader Tips</td>
			<td style="width:189px;">Eppendorf</td>
			<td style="width:287px;">C2554691</td>
			<td>Backfilling needle loading tips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Tissue Culture Dishes</td>
			<td style="width:189px;">Falcon</td>
			<td>25383-103</td>
			<td>Sprouting corn /150 x 25 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisherbrand&#160;Quantitative-Grade Filter Paper Circles</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td>S47576C</td>
			<td>Making agar dish for egg-lay/9cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sparkleen</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td style="width:287px;">04-320-4</td>
			<td>Wash dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Plain Microscope Slides</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td style="width:287px;">12-549-3</td>
			<td>Holding filter paper and eggs for microinjection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Western Corn Rootworm w/o Pollen Substitute</td>
			<td style="width:189px;">Frontier Agricultural Sciences&#160;</td>
			<td>F9766B</td>
			<td>WCR adult artificial diet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cotton Balls, Large</td>
			<td>Genesee Scientific</td>
			<td>51-101</td>
			<td>Close the flask</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Globe Scientific 3.0 mL Small Bulb Transfer Pipettes</td>
			<td>Globe Scientific</td>
			<td>137035</td>
			<td>Collect and transfer eggs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica M165 FC Fluorescence Stereomicroscope&#160;</td>
			<td style="width:189px;">Leica</td>
			<td>M165 FC&#160;</td>
			<td>WCR screening</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DsRed Filter Set for Fluorescence Stereomicroscope</td>
			<td style="width:189px;">Leica</td>
			<td>DSR</td>
			<td>Excitation filter: 510-560 nm, emission filter: 590-650 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGFP filter</td>
			<td style="width:189px;">Leica</td>
			<td>GFP2</td>
			<td>Excitation filter: 440&#8211;520 nm, emission filter: 510 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BugDorm</td>
			<td>MegaView Science</td>
			<td>DP1000_5P</td>
			<td>Cage for adult colony</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Miniature Paint Brush</td>
			<td style="width:189px;">MyArtscape</td>
			<td>MAS-102-MINI</td>
			<td>Liner 2/0&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Joystick Micromanipulator</td>
			<td style="width:189px;">Narishige</td>
			<td style="width:287px;">MN-151</td>
			<td>Micromanipulator and needle holder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">Microscope XY Stage</td>
			<td style="width:189px;">Olympus</td>
			<td style="width:287px;">265515</td>
			<td style="width:339px;">Microscope stage (adapted for use with stereoscope)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Grade 90 Cheesecloth</td>
			<td style="width:189px;">Online Fabric Store</td>
			<td>CHEE90</td>
			<td>For egg-lay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic paraffin film&#160;</td>
			<td style="width:189px;">Pechiney Plastic Packaging</td>
			<td>PM-996</td>
			<td>Seal agar dish after microinjection/Roll size 4 in. x 125 ft</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Percival Incubator</td>
			<td style="width:189px;">Percival&#160;</td>
			<td style="width:287px;">I41VLH3C8</td>
			<td>WCR growing chamber (insect rearing incubator)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Narrow Mouth Erlenmeyer Flasks</td>
			<td style="width:189px;">VWR</td>
			<td>4980-300-PK</td>
			<td>Water container for adult colony&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Griffin Low Form Beakers</td>
			<td style="width:189px;">VWR</td>
			<td>1000-600-PK</td>
			<td>For egg wash</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Braided Cotton Rolls</td>
			<td>Richmond Dental Cotton Co.</td>
			<td>605-3599</td>
			<td>Use in water supply for adult colony&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dishes (35x10mm)</td>
			<td>SIGMA</td>
			<td>CLS430588-500EA</td>
			<td>For diet plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenol Red</td>
			<td>Sigma</td>
			<td style="width:287px;">143-78-8</td>
			<td>Microinjection buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laser-based Micropipiette Puller</td>
			<td>Sutter Instrument</td>
			<td>P-2000/G</td>
			<td>Needle puller/Heat = 335, FIL = 4, VEL = 40, DEL = 200, PUL = 100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Premium Topsoil</td>
			<td>The Scotts Company</td>
			<td style="width:287px;">71130758</td>
			<td>Soil for cron growing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reloc Zippit 2 Mil Zipper Bags</td>
			<td>United States Plastic Corporation</td>
			<td>48342</td>
			<td>Bag agar dish after microinjection/5x7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dishes (100x15m)</td>
			<td style="width:189px;">VWR</td>
			<td style="width:287px;">89038-968</td>
			<td>Making agar dish for egg-lay/ 100 x 15 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 oz Containers</td>
			<td style="width:189px;">Webstaurantstore</td>
			<td>128E506</td>
			<td>WCR single pair mating chamber&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">38 oz Containers</td>
			<td style="width:189px;">Webstaurantstore</td>
			<td>128NC888</td>
			<td>Larvae rearing box/38 oz</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ChoiceHD 16 oz. Microwavable Translucent Plastic Deli Container</td>
			<td style="width:189px;">Webstaurantstore</td>
			<td>128HRD16</td>
			<td>Larvae rearing box/16 oz</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Microinjection Scope&#160;</td>
			<td style="width:189px;">Wild Heerbrugg</td>
			<td style="width:287px;">WILD-M8</td>
			<td style="width:339px;">Microinjection scope outfited with an XY stage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100F-4</td>
			<td>Microinjection needles</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">Adult Western Corn Rootworms</td>
			<td style="width:189px;">North Centeral Aqricultural reasearch Laboratory</td>
			<td style="width:287px;">Non-diapase strain</td>
			<td>Request from Dr. Bryan Wade French&#39;s lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Plasmid DNA Midi Kit</td>
			<td style="width:189px;">Qiagen</td>
			<td>12143</td>
			<td>Purification of injection-ready plasmid DNAs</td>
		</tr>
	</tbody>
</table>

microinjection of western corn rootworm, diabrotica virgifera virgifera, embryos for germline transformation, or crispr/cas9 genome editing

Vestlige mais rootworm (WCR) er en viktig pest av korn og er kjent for sin evne til å raskt tilpasse seg pest management strategier. Selv om RNA-interferens (RNAi) har vist seg for å være et kraftig verktøy for å studere WCR biologi, har sine begrensninger. Spesielt RNAi selv er forbigående (dvs. ikke fører til langsiktig Mendelian arv av tilknyttede fenotypen), og det krever å vite DNA sekvensen av målet genet. Sistnevnte kan være begrensende hvis fenotypen rundt kontrolleres av dårlig bevart eller selv romanen gener, fordi identifisere nyttig mål vil være utfordrende, om ikke umulig. Antallet verktøy i WCRS genomisk verktøykassen bør derfor utvides ved utviklingen av metoder som kan brukes til å opprette stabil mutant stammer og aktivere sekvens-uavhengige undersøkelser av WCR genomet. Her, detalj vi metodene som brukes til å samle og microinject precellular WCR embryo med nucleic syrer. Mens protokollene beskrevet her er rettet mot etableringen av transgene WCR, kan CRISPR/Cas9-genomet redigering også utføres ved hjelp av samme protokoller, med den eneste forskjellen er sammensetningen av løsningen injiseres i embryoer.

En viktig faktor når du utvikler denne protokollen ble embryonale utvikling ved microinjection. Spesielt krever germline transformasjon microinjection av DNAs før embryonale cellularization. Dette er fordi DNAs ikke kan enkelt krysse cellemembraner. Forsøk å visualisere stadier av WCR embryonale utvikling ved hjelp av en kjernefysisk flekk var mislykket fordi dechorionation av WCR embryo egentlig oppløser alle beskyttende membraner ansvarlig for integriteten til fosteret. Men ble den...

Watch this Scientific Journal Video about Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing at JoVE.com

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Microinjection Western mais Rootworm, Diabrotica virgifera virgifera, embryo for Germline transformasjon eller CRISPR/Cas9 genomet redigering

Her presenterer vi protokoller for innsamling og microinjecting precellular vestlige korn rootworm embryo for å utføre funksjonelle-genomisk analyser som germline transformasjon og CRISPR/Cas9-genomet redigering.

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although ...

Research

JoVE Journal

Bioengineering

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Viral Nanoparticles for In vivo Tumor Imaging

Viral Nanoparticles for In vivo Tumor Imaging

Viral Nanopartikler for<em&gt; In vivo</em&gt; Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

Mikroinjeksjon Wound-analysen og<em&gt; In vivo</em&gt; Lokalisering av epidermal Wound Response Reportere i<em&gt; Drosophila</em&gt; Embryo.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Hydrogel Nanoparticle Høsting av plasma eller urin for å påvise lav overflod Proteiner

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Effektiv generasjon og redigering av mater-gratis IPSCs fra menneskelige bukspyttkjertelen celler ved hjelp av CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

Produksjon av genmodifiserte Golden syrer Hamsters ved Pronuclear injeksjon av CRISPR/Cas9 komplekset

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. ...

Ultra-long Read Sequencing for Whole Genomic DNA Analysis

Svært lang lese sekvenser for hele genomisk DNA-analyse

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes ...

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

CRISPR/Cas12a multiplex Genova redigering av Saccharomyces cerevisiae og opprettelse av gjær pixel art

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) ...

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

Måle spin-gitter avslapning magnetisk felt avhengighet av Hyperpolarized [1-13C] pyruvat

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various ...

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

Mikroinjeksjon av maisplanthopper, Peregrinus maidis, embryoer for CRISPR/Cas9-genomredigering

The corn planthopper, Peregrinus maidis, is&#160;a pest of maize and a vector of several maize viruses. Previously published methods describe the ...

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Flytende kromatografi kombinert med brytningsindeks eller massespektrometrisk deteksjon for metabolittprofilering i lysatebaserte cellefrie systemer

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the ...