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 arbejde blev støttet af en bevilling fra Monsanto majs-rodorm viden Research Program, give nummer AG/1005 (til MDL og YC) og start-up midler til MDL fra NCSU. FC blev støttet af tilskud fra Monsantos majs-rodorm viden Research Program (AG/1005) og National Science Foundation, tildele nummer MCB-1244772 (MDL). Forfatterne erklærer ikke konkurrerende interesser. FC og MDL udtænkt og designet eksperimenter; FC, PW og SP udførte eksperimenter; FC og MDL analyseret resultaterne; og FC, NG og MDL skrev manuskriptet. Vi takker Teresa O'Leary, William Klobasa og Stephanie Gorski for deres ekspertbistand i screening VIF. Vi takker også Dr. Wade French (USDA-ARS, North Central landbrugs forskningslaboratorium, Brookings, SD) for overførsel af æg at etablere lab kolonier og leverer opdræt protokoller.

1. koloni-niveau opdræt af VIF voksne
<ol><li>Få en tilstrækkelig mængde af VIF voksne (500-1.000) fra en pålidelig virksomhed eller forskning laboratorium (Se Tabel af materialer for eksempel) og placere i en 30 cm3 bur. Bemærk: Brug af en ikke-diapausing stamme er stærkt anbefales.</li>
	<li>Forberede VIF kunstig kost efter producentens protokol og hæld et 1 cm tykt lag i en 38 ounce container (Se Tabel af materialer). Efter blanding, skal du gemme ubrugte kost ...

<ol>
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Selvom mikroinjektion af VIF med dsRNAs med henblik på RNAi har været rapporteret9, dette er den første protokol til at fastlægge bedste praksis for microinjecting precellular VIF embryoner, en kritisk proces for at gennemføre kønscelleoverførsel transformation og/eller CRISPR/Cas9 genom-redigering i denne art. Vellykket mikroinjektion af VIF embryoner er afhængig af mange faktorer, som er transformation effektivitet. Diskuteret nedenfor er nogle af de store spørgsmål påvirker resultate...

Majs-rodorm (VIF), Diabrotica virgifera virgifera, er et vigtigt skadedyr af majs1. Interessant, VIF synes at overvinde kontrol foranstaltninger hurtigere end mest landbrugs skadedyr, da de ikke kun tilpasse fysiologisk men også behaviorally2,3,4,5, 6. i det sidste årti, RNA-interferens (RNAi), en kraftfuld funktionelle genomisk værktøj, er blevet undersøgt som en potentiel kontrol metode for VIF7,8, og er også blevet brugt som et middel til at studere gen funktion9. Men mens RNAi udføres ofte ved mikroinjektion af dobbelt-strenget RNA (dsRNA) i andre arter, injektion-baserede RNAi er sjældne i VIF. I virkeligheden er der kun få rapporter om RNAi via mikroinjektion af dsRNA i VIF9. Årsagen er, at VIF kan nå høje niveauer af genet knockdown via indtagelse af dsRNA7,10, selv tillader undersøgelse af embryonale effekter ved at fodre dsRNA mor11. Mens denne metode gør VIF et glimrende emne for genomisk funktionalanalyse via RNAi, er det aftaget fremskridt på udvikling af metoder til embryonale mikroinjektion i denne art.
Trods effekt af RNAi er der et par ulemper. For eksempel, reagere ikke alle gener lige så at RNAi. Denne variation kan gøre fortolkningen af en funktionel genomforskning assay vanskeligere. Også, RNAi er forbigående og genererer ikke arvelige mutationer. På den anden side kan kønscelleoverførsel transformation, en anden funktionel genomisk værktøj, generere arvelige mutationer via indsættelse af en markant varemærkeregistreringer element i genom12,13. Dette gør kønscelleoverførsel transformation et fremragende værktøj til at skabe mutantstammen til brug i langsigtede genetiske undersøgelser. Transformationen kan også bruges til at redde en mutation ved at levere en funktionel kopi af genet til en mutant genom14. Derudover er kønscelleoverførsel transformation hjørnestenen i en bred vifte af molekylær genetiske teknikker. Ud over udskære og/eller redning genfunktion, kan kønscelleoverførsel transformation bruges til forstærker fældefangst15, gen fældefangst16, Gal4-baserede Ektopisk udtryk17og genome-wide mutagenese18.
For nylig er blevet udviklet værktøjer, der aktiverer sofistikerede genom-redigering. Disse værktøjer omfatter transskription Activator-Like effektor nukleaser (TALEN) og CRISPR/Cas9-nukleasen system19,20. Fordelen ved disse nye værktøjer er, at de tilbyder forskere evne til at fremkalde en dobbelt-strenget DNA pause på næsten ethvert sted inden for næsten alle genom. Når induceret, disse pauser kan repareres via enten ikke-homologe ende sammenføjning, som kan præsentere sletninger eller indrykninger i målrettede genet, eller homologi-instrueret reparation, som i tilstedeværelse af en manipuleret konstruktion, kan katalysere den udskiftning af hele gener eller genetiske regioner21,22. Disse metoder kræver dog mikroinjektion af DNA, RNA og/eller protein i meget ung embryoner.
Vigtigere, i modsætning til dsRNAs anvendes til RNAi, nukleinsyrer og proteiner anvendes for kønscelleoverførsel kan ikke transformation og genom-redigering nemt krydse cellemembraner. Derfor skal mikroinjektion af plasmid DNAs, mRNAs og/eller proteiner finde sted under syncytial blastoderm fase (dvs. før den insekt embryo cellularizes). Dette gør timingen af injektioner en kritisk faktor. For eksempel i Drosophila melanogasterskal embryoner sprøjtes inden for to timer efter æg lå12. Derfor, udvikling af en vellykket embryonale mikroinjektion protokol for VIF skal tage hensyn de bedste betingelser for kvindelige æglægning, samt den bedste metode til at indsamle tilstrækkelige mængder af precellular embryoner.
Et forsøg på at bringe en bred vifte af molekylær genetiske værktøjer til at bære på VIF biologi kræver udvikling af metoder til indsamling og microinjecting precellular VIF embryoner. Her leverer vi detaljerede instruktioner, sammen med tips og tricks, til at hjælpe andre anvende transformation-baserede teknikker i VIF forskning. Ud over at udvide transgene teknologier til VIF til brug i funktionelle genomisk undersøgelser, aktiverer disse teknikker også kraftfulde nye pest control strategier såsom gen drev23,24 skal testes i denne økonomisk vigtige skadedyr.

<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

Majs-rodorm (VIF) er en vigtige skadedyr af majs og er kendt for sin evne til hurtigt tilpasse sig til pest forvaltningsstrategier. Selvom RNA-interferens (RNAi) har vist sig for at være et effektivt redskab til at studere VIF biologi, har den sine begrænsninger. Specifikt, RNAi selv er forbigående (dvs. ikke resulterer i langsigtede mendelske arv af de tilknyttede fænotype), og det kræver at vide DNA-sekvens af target-genet. Sidstnævnte kan være begrænsende hvis Fænotypen af interesse er kontrolleret af dårligt bevarede, eller endda nye gener, fordi at identificere nyttige mål ville være udfordrende, hvis ikke umuligt. Antallet af værktøjer i VIFS genomisk værktøjskasse bør derfor udvides ved udviklingen af metoder, der kunne bruges til at skabe stabile mutantstammen og aktiverer sekvens-uafhængige undersøgelser af VIF genom. Heri, detalje vi de metoder, der anvendes til at indsamle og microinject precellular VIF embryoner med nukleinsyrer. Mens de protokoller er beskrevet heri er rettet mod oprettelsen af transgene VIF, kan CRISPR/Cas9-genom-redigering også udføres ved hjælp af de samme protokoller, med den eneste forskel er sammensætningen af opløsningen injiceres embryoner.

En vigtig overvejelse ved udviklingen af denne protokol var fase af fosterudviklingen ved mikroinjektion. Specifikt, kræver kønscelleoverførsel transformation mikroinjektion af DNAs før embryonale cellularization. Dette er fordi DNAs let kan krydse cellemembraner. At visualisere stadier af VIF embryonale udvikling ved hjælp af en nuklear pletten var kundescenario fordi dechorionation af VIF embryoner hovedsagelig opløser alle beskyttende membraner ansvarlig for integriteten af embry...

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

Mikroinjektion af Western majs-rodorm Diabrotica virgifera virgifera, embryoner for kønscelleoverførsel Transformation, eller CRISPR/Cas9 genom-redigering

Her præsenterer vi protokoller for indsamling og microinjecting precellular vestlige majs rodorm embryoner med henblik på udførelse af funktionelle-genomisk assays kønscelleoverførsel transformation og CRISPR/Cas9-genom-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

Virale 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.

Mikroinjektion Wound Assay og<em&gt; In vivo</em&gt; Lokalisering af epidermale Wound Respons Journalister i<em&gt; Drosophila</em&gt; Embryoner.

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 Nanopartikel Høst af plasma eller urin til påvisning af lav tæthed 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 Generation og redigering af Feeder-gratis IPSCs fra pancreas celler ved hjælp af 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

Produktion af gensplejsede gyldne syriske hamstere af Pronuclear injektion af 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

Ultra-lang Læs sekventering 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 Genome redigering af Saccharomyces cerevisiae og skabelsen af gær pixel kunst

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

Måling af spin-Lattice afslapning magnetisk felt afhængighed af Hyperpolariseret [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

Mikroinjektion af majsplantehoppe, Peregrinus maidis, embryoner til 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

Flydende kromatografi koblet til brydningsindeks eller massespektrometrisk detektion til metabolitprofilering i lysatbaserede cellefrie systemer

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