Name: functional characterization of carboxylesterases in insecticide resistant house flies, musca domestica
Uploaded: 2018-08-23T14:30:00
Description: Watch this Scientific Journal Video about Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica at JoVE.com

1. weergave en isolatie van Target proteïnen met een Insect cel Baculovirus-gemedieerde expressie systemen
<ol><li>Gerichte kloon blunt-ended PCR producten van doel eiwitten uit huis vliegt.<ol>
<li>Ontwerp PCR inleidingen van groen fluorescent proteïne (GFP) en het huis vliegen MdαE7 gen gebaseerd is op hun reeksen en de bijzondere noden van de gekozen vector (tabel 1).</li>
		<li>Een thermostable, corrigeren polymerase van DNA- en grondlagen uit stap 1.1.1 gebruiken voor het uitvoeren van een 150 ...

<ol>
	<li>Scott, J. G., 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=Insecticide+resistance+in+house+flies+from+the+United+States:+Resistance+levels+and+frequency+of+pyrethroid+resistance+alleles.">Insecticide resistance in house flies from the United States: Resistance levels and frequency of pyrethroid resistance alleles.</a> Pesticide Biochemistry and Physiology. 107 (3), 377-384 (2013).</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=A+whole+transcriptomal+linkage+analysis+of+gene+co-regulation+in+insecticide+resistant+house+flies,+Musca+domestica.">A whole transcriptomal linkage analysis of gene co-regulation in insecticide resistant house flies, Musca domestica.</a> BMC Genomics. 14, 803 (2013).</li><li>Liu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insecticide+resistance+in+mosquitoes:+impact,+mechanisms,+and+research+directions.">Insecticide resistance in mosquitoes: impact, mechanisms, and research directions.</a> Annual Review of Entomology. 60, 537-559 (2015).</li><li>Grigoraki, L., 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=Transcriptome+profiling+and+genetic+study+reveal+amplified+carboxylesterase+genes+implicated+in+temephos+resistance,+in+the+Asian+tiger+mosquito+Aedes+albopictus.">Transcriptome profiling and genetic study reveal amplified carboxylesterase genes implicated in temephos resistance, in the Asian tiger mosquito Aedes albopictus.</a> e0003771. 9, e0003771 (2015).</li><li>Grigoraki, L., 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=Carboxylesterase+gene+amplifications+associated+with+insecticide+resistance+in+Aedes+albopictus:+Geographical+distribution+and+evolutionary+origin.">Carboxylesterase gene amplifications associated with insecticide resistance in Aedes albopictus: Geographical distribution and evolutionary origin.</a> PLOS Neglected Tropical Diseases. 11, e0005533 (2017).</li><li>Wheelock, C., Shan, G., Ottea, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+carboxylesterases+and+their+role+in+the+metabolism+of+insecticides.">Overview of carboxylesterases and their role in the metabolism of insecticides.</a> Journal of Pesticide Science. 30, 75-83 (2005).</li><li>Feng, X., Li, M., Liu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carboxylesterase+genes+in+pyrethroid+resistant+house+flies,+Musca+domestica.">Carboxylesterase genes in pyrethroid resistant house flies, Musca domestica.</a> Insect Biochemistry and Molecular Biology. 92, 30-39 (2018).</li><li>Mosmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+colorimetric+assay+for+cellular+growth+and+survival:+application+to+proliferation+and+cytotoxicity+assays.">Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.</a> J Immunol Methods. 65 (1-2), 55-63 (1983).</li><li>Jarvis, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus-insect+cell+expression+systems.">Baculovirus-insect cell expression systems.</a> Methods in Enzymology. 463, 191-222 (2009).</li><li>Berger, I., Fitzgerald, D. J., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus+expression+system+for+heterologous+multiprotein+complexes.">Baculovirus expression system for heterologous multiprotein complexes.</a> Nature Biotechnology. 22 (12), 1583 (2004).</li><li>Gong, Y., Li, T., Feng, Y., Liu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+function+of+two+P450s,+CYP9M10+and+CYP6AA7,+in+the+permethrin+resistance+of+Culex+quinquefasciatus.">The function of two P450s, CYP9M10 and CYP6AA7, in the permethrin resistance of Culex quinquefasciatus.</a> Scientific Reports. 7 (1), 587 (2017).</li><li>Cao, C. W., Zhang, J., Gao, X. W., Liang, P., Guo, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+carboxylesterase+gene+associated+with+organophosphorous+insecticide+resistance+in+cotton+aphids,+Aphis+gossypii+(Glover).">Overexpression of carboxylesterase gene associated with organophosphorous insecticide resistance in cotton aphids, Aphis gossypii (Glover).</a> Pesticide Biochemistry and Physiology. 90 (3), 175-180 (2008).</li><li>Zhang, L., Gao, X., Liang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-cypermethrin+resistance+associated+with+high+carboxylesterase+activities+in+a+strain+of+house+fly,+Musca+domestica+(Diptera:+Muscidae).">Beta-cypermethrin resistance associated with high carboxylesterase activities in a strain of house fly, Musca domestica (Diptera: Muscidae).</a> Pesticide Biochemistry and Physiology. 89, 65-72 (2007).</li><li>Van Meerloo, J., Kaspers, G. J., Cloos, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+sensitivity+assays:+the+MTT+assay.">Cell sensitivity assays: the MTT assay.</a> Cancer cell culture. , 237-245 (2011).</li><li>Stockert, J. C., Bl&#225;zquez-Castro, A., Ca&#241;ete, M., Horobin, R. W., Villanueva, &. #. 1. 9. 3. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MTT+assay+for+cell+viability:+Intracellular+localization+of+the+formazan+product+is+in+lipid+droplets.">MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets.</a> Acta Histochemica. 114 (8), 785-796 (2012).</li><li>Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypan+blue+exclusion+test+of+cell+viability.">Trypan blue exclusion test of cell viability.</a> Current Protocols in Immunology. , (2001).</li><li>Riss, T. L., Moravec, R. A., Niles, A. L., Duellman, S., Benink, H. A., Worzella, T. J., Minor, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+viability+assays.">Cell viability assays.</a> Assay Guidance Manual. , (2013).</li><li>Wheelock, C. E., Shan, G., Ottea, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+carboxylesterases+and+their+role+in+the+metabolism+of+insecticides.">Overview of carboxylesterases and their role in the metabolism of insecticides.</a> Journal of Pesticide Science. 30 (2), 75-83 (2005).</li><li>Li, X., Schuler, M. A., Berenbaum, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+metabolic+resistance+to+synthetic+and+natural+xenobiotics.">Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics.</a> Annual Review of Entomology. 52, 231-253 (2007).</li><li>Nakamura, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vitro+metabolism+of+a+pyrethroid+insecticide,+permethrin,+and+its+hydrolysis+products+in+rats.">The in vitro metabolism of a pyrethroid insecticide, permethrin, and its hydrolysis products in rats.</a> Toxicology. 235 (3), 176-184 (2007).</li><li>Kruger, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bradford+method+for+protein+quantitation.">The Bradford method for protein quantitation.</a> The protein protocols handbook. , 15-21 (2002).</li><li>Macauley-Patrick, S., Fazenda, M. L., McNeil, B., Harvey, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterologous+protein+production+using+the+Pichia+pastoris+expression+system.">Heterologous protein production using the Pichia pastoris expression system.</a> Yeast. 22 (4), 249-270 (2005).</li><li>Berger, I., Fitzgerald, D. J., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus+expression+system+for+heterologous+multiprotein+complexes.">Baculovirus expression system for heterologous multiprotein complexes.</a> Nature Biotechnology. 22 (12), 1583 (2004).</li><li>Terpe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+bacterial+expression+systems+for+heterologous+protein+production:+from+molecular+and+biochemical+fundamentals+to+commercial+systems.">Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems.</a> Applied Microbiology and Biotechnology. 72 (2), 211 (2006).</li><li>Bulter, T., 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=Functional+expression+of+a+fungal+laccase+in+Saccharomyces+cerevisiae+by+directed+evolution.">Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution.</a> Applied Microbiology and Biotechnology. 69 (2), 987-995 (2003).</li><li>Stepanenko, A. A., Dmitrenko, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pitfalls+of+the+MTT+assay:+Direct+and+off-target+effects+of+inhibitors+can+result+in+over/underestimation+of+cell+viability.">Pitfalls of the MTT assay: Direct and off-target effects of inhibitors can result in over/underestimation of cell viability.</a> Gene. 574 (2), 193-203 (2015).</li></ol>

In de afgelopen decennia hebben heterologe expressiesystemen wijd gebruikt om te uiten en isoleren van grote hoeveelheden eiwitten, waardoor biochemische en functionele vastberadenheid en karakterisering van enzymen in vitro. Tot op heden zijn verscheidene verschillende modelsystemen waaronder Escherichia coli, Sacccharomyces cerevisiae, Pichia pastorisen Spodoptera frugiperda aangepast voor recombinant eiwit expressie, en de keuze van de in vitro systeem is van cruci...

Insecticide resistentie is momenteel een groot probleem voor huis vliegen controle wereldwijd1,2. Inspanningen om het mechanisme van insecticide resistentie vergemakkelijkt beter begrip van deze kwestie en bieden daardoor nieuwe strategieën om effectief voorkomen of minimaliseren van de verspreiding van resistentie ontwikkeling3. Carboxylesterases, hebben als een van de grote Detoxificatie-enzymen, aangetrokken veel aandacht voor hun rollen in vastleggen en insecticiden in verschillende insecten4,5,6hen te metaboliseren. Onze vorige studie geconstateerd meerdere carboxylesterases in huis vliegt en hun expressie niveaus waren niet alleen constitutively omhoog-geregeld in de resistente stam van de ALHF maar kunnen ook geïnduceerde naar hogere niveaus in reactie op permethrin behandelingen7 . De functionele karakteristieken van deze carboxylesterase genen in de stofwisseling van insecticiden moeten echter nog worden onderzocht.
Sinds het eerste verslag in de vroege jaren 19808, heeft een baculovirus-gemedieerde buitenlandse gen expressie systeem wijd uitgeoefend vanwege de hoge eiwit productie-efficiëntie en het eukaryotische eiwit verwerking mogelijkheden9. Deze binaire systeem bestaat uit twee essentiële elementen: de geconstrueerde recombinante baculovirus leveren van vreemde genen in de cellen van de gastheer, en de grootschalige uitdrukking van interesse eiwitten door cellen besmet door recombinant baculovirus. In de afgelopen decennia, het baculovirus gemedieerde cel expressie systeem is wijd verbeid gebruikt voor de productie van recombinante eiwitten, variërend van cytosolische enzymen aan membraan-gebonden eiwitten in insect duizenden en zoogdier cellen10. Onze vorige studie heeft met succes meerdere CYP450 enzymen in insect cellen van de Sf9 met dit systeem11. In deze studie, we gebouwd een carboxylesterase-recombinant baculovirus om insecten Sf9 cellen infecteren, onderzocht de tolerantie van de cel voor verschillende permethrin behandelingen, en grootschalige uitgedrukt carboxylesterase eiwitten in vitro voor functionele verkenning. In plaats van het onderzoeken van meerdere carboxylesterase isozyme mengsels van insecten homogenates zoals aangenomen door de vorige studies12,13, dit insect cel baculovirus-gemedieerde expressie systeem maakt het mogelijk de specifieke expressie en isolatie van gerichte eiwitten voor betere karakterisering van de biochemische en structurele eigenschappen.
De tetrazolium zout gebaseerde bepaling (MTT) is een high-throughput colorimetrische methode ontwikkeld en geoptimaliseerd voor het meten van de levensvatbaarheid van de cellen. Deze test is gebaseerd op het mechanisme dat alleen levende cellen hen metaboliseren het geel uitdrukkingsloos MTT reagens aan een donkere paarse gekleurde formazan neerslag, die kan worden colorimetrisch geanalyseerd kunnen nadat opgelost in organische oplosmiddelen14, 15. Verschillende nauwkeuriger maar tijdrovende methoden, zoals Trypan blauwe uitsluiting en de thymidine titratie assay16,17, ontwikkeld in de afgelopen jaren. De MTT cel-gebaseerde bepaling is echter nog steeds erkend als de meest snelle en gemakkelijk bediende methode voor snel opsporen levensvatbaarheid van de cellen. Hier, gebruiken we de MTT-test om te verkennen de tolerantie van de cel tegen insecticide behandelingen. De verbeterde tolerantie van cellen wanneer geïnfecteerd met carboxylesterase recombinante baculovirus sterk ondersteunt de metabole functies van carboxylesterases tot insecticiden, die op zijn beurt suggereert hun betrokkenheid in insecticide resistentie.
Bovendien werd een in vitro metabole test ook uitgevoerd in deze studie. Vergeleken met de algemene carboxylesterase tests waarmee gemeenschappelijke substraten zoals α-napthyl acetaat (α-nvt) en β-naphthyl-acetaat (β-nvt) weerspiegelen Hydrolytische activiteiten van carboxylesterases, wordt de in vitro metabole studie beschouwd als een accurate manier direct meten activiteiten van carboxylesterases naar insecticiden18. Deze methode heeft met succes gewerkt in verschillende insecten te karakteriseren van meerdere cytochroom P450s i.s.m. insecticide resistentie11,19,20. Echter, deze methode heeft nog niet vereffend in carboxylesterase studies. Met de beschikbaarheid van carboxylesterase-eiwitten geproduceerd door baculovirus-gemedieerde expressie systeem, we kunnen het uitvoeren van een in vitro metabole studie van carboxylesterases naar permethrin, die verder harde bewijzen voor de betrokkenheid kan bieden van carboxylesterases in de toekenning van pyrethroïde weerstand in huis vliegt.

<table>
	<tbody>
		<tr>
			<td>Q5 High-Fidelity DNA Polymerase</td>
			<td>New England Biolabs inc.</td>
			<td>M0491L</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>QIAGEN</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>pENTR/D-TOPO Cloning Kit, with One Shot TOP10 Chemically Competent E. coli</td>
			<td>Invitrogen by life technology</td>
			<td>K240020</td>
			<td>S.O.C medium and universal M13 sequence primers were included in this kit.</td>
		</tr>
		<tr>
			<td>PureLink HiPure Plasmid Miniprep Kit</td>
			<td>Invitrogen by life technology</td>
			<td>K210002</td>
			<td></td>
		</tr>
		<tr>
			<td>Gateway LR Clonase II Enzyme mix for BaculoDirectTM Kits</td>
			<td>Invitrogen by life technology</td>
			<td>11791-023</td>
			<td></td>
		</tr>
		<tr>
			<td>BaculoDirect C-Term Linear DNA Transfection Kit</td>
			<td>Invitrogen by life technology</td>
			<td>12562-019</td>
			<td>Cellfectin transfection reagent and ganciclovir were included in this kit</td>
		</tr>
		<tr>
			<td>pENTR-CAT plasmid</td>
			<td>Invitrogen by life technology</td>
			<td></td>
			<td>Included in BaculoDirect C-Term Linear DNA Transfection Kit, concentration: 0.5 ug/uL</td>
		</tr>
		<tr>
			<td>Heat inactivated Fetal Bovine Serum, Certified</td>
			<td>Gibco by Life Technologies</td>
			<td>10082-139</td>
			<td></td>
		</tr>
		<tr>
			<td>Sf9 cells in Sf-900 III SFM</td>
			<td>Gibco by Life Technologies</td>
			<td>12659017</td>
			<td></td>
		</tr>
		<tr>
			<td>Insect Cell-PE LB Insect Cell Protein Extraction &#38; Lysis Buffer</td>
			<td>G Biosciences by A Geno Technology Inc</td>
			<td>786-411</td>
			<td></td>
		</tr>
		<tr>
			<td>Sf-900 III SFM (1&#215;) Serum Free Medium Complete</td>
			<td>Gibco by Life Technologies</td>
			<td>12658-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Grace&#39;s Insect Medium, unsupplemented</td>
			<td>Gibco by Life Technologies</td>
			<td>11595030</td>
			<td></td>
		</tr>
		<tr>
			<td>Permethrin (isomers) analytical standard</td>
			<td>SUPELCO by Solutions WithinTM</td>
			<td>442748</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol (analytical graded)</td>
			<td>Sigma-Aldrich</td>
			<td>67-56-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile (analytical graded)</td>
			<td>Sigma-Aldrich</td>
			<td>75-05-8</td>
			<td></td>
		</tr>
		<tr>
			<td>GHP Acrodisc 25 mm Syringe Filters with 0.45 &#956;m GHP Membrane (HPLC Certified)</td>
			<td>Pall Life Sciences</td>
			<td>21890388</td>
			<td></td>
		</tr>
		<tr>
			<td>Alliance Waters 2695 HPLC System</td>
			<td>Waters</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T100 Thermal Cycle</td>
			<td>Bio-Rad Laboratories Inc.</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop 2000/2000c Spectrophotometers</td>
			<td>ThermoFisher Scientific</td>
			<td>ND2000CLAPTOP</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytation 5 Cell Imaging Multi-Mode Reader</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

functional characterization of carboxylesterases in insecticide resistant house flies, musca domestica

Carboxylesterase-gemedieerde metabolisme wordt beschouwd als een belangrijke rol spelen in insecticide resistentie bij verschillende insecten. Meerdere carboxylesterase genen bleken omhoog-geregeld in de resistente huis vliegen stam, terwijl hun rol in de overdracht van het insecticide resistentie bleef om te worden onderzocht. Hier, ontwierpen we een protocol voor de functionele karakterisering van carboxylesterases. Drie voorbeeld experimenten worden gepresenteerd: (1) expressie en isolatie van proteïnen van de carboxylesterase door een insect van baculovirus-gemedieerde Spodoptera frugiperda (Sf9) systeem voor de expressie van de cel; (2) een cel-gebaseerde MTT (3-[5-dimethykthiazol-2-yl 4] -2, 5-diphenyltetrazolium bromide) cytotoxiciteit assay voor het meten van de tolerantie van insect cellen tot verschillende permethrin behandelingen; en (3) in vitro metabole studies om te verkennen de metabole mogelijkheden van carboxylesterases naar permethrin. Het carboxylesterase gen MdαE7 werd gekloond uit een resistente huis vliegen stam ALHF en gebruikt voor de constructie van een recombinant baculovirus voor Sf9 cellen infectie. De viabilities van de cel tegen verschillende permethrin behandelingen werden gemeten met de MTT-test. De verbeterde cel tolerantie van de experimentele groep (MdαE7-recombinant baculovirus geïnfecteerde cellen) vergeleken met die van de controlegroepen (CAT-recombinant baculovirus geïnfecteerde cellen en GFP-recombinant baculovirus geïnfecteerde cellen) naar permethrin behandelingen voorgesteld de mogelijkheden van MdαE7 in de stofwisseling van insecticiden, waardoor de bescherming van cellen tegen chemische schade. Daarnaast werden carboxylesterase eiwitten uitgedrukt in insect cellen van de Sf9 en om uit te voeren van een metabole studie in vitro geïsoleerd. Onze resultaten aangegeven een aanzienlijke in vitro metabole efficiëntie van MdαE7 naar permethrin, direct de betrokkenheid van carboxylesterases in de stofwisseling van insecticiden die aangeeft en dus de toekenning van insecticide resistentie in huis vliegt.

De levensvatbaarheid van de cellen naar verschillende permethrin behandelingen (MTT assay)
De cytotoxiciteit van permethrine werd onderzocht in MdαE7-recombinant baculovirus besmet Sf9 cellen (experimentele groep) en CAT-recombinant baculovirus (verstrekt door baculovirus besmet kit) geïnfecteerde cellen (controlegroepen). De verbeterde cel toleranties aan permethrine in MdαE7 uitdrukken cellen sterk ondersteunen de metabolische...

Watch this Scientific Journal Video about Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica at JoVE.com

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Hier presenteren we een protocol om te produceren huis vliegen carboxylesterase eiwitten in vitro met een baculovirus gemedieerde insect cel expressie systeem en later functioneel karakteriseren van hun rollen in metaboliseringssysteem permethrin, daarmee overdracht van pyrethroïde weerstand door het uitvoeren van cel-gebaseerde MTT assay en in vitro metabole studies.

Functionele karakterisering van Carboxylesterases in Insecticide resistente huis vliegt, Musca Domestica

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were ...

This method can help the key questions in the field of insect toxicology, such as, if all express the metabolic detoxification genes are involved in insecticide resistance? The main advantage of this technique is that it allows to produce larger scales of proteins, and characterize the function of these proteins in metabolizing of insecticides in vitro. Demonstrating the procedures, will be Xuechun Feng, a grad student in my laboratory.Xuechen's PhD research is focused on the characterization of function of carboxyl esterase in parasite resistance of house flies, Musca domestica. To begin, mix one microliter of GFP entry plasma DNA, with five microliters of a commercially available C-term linear DNA in 250 microliter PCR tubes. Next, add two microliters of commercially available lambda recombination mix to each of the PCR tubes.Gently mix the tubes, and incubate them at 25 degrees Celsius overnight. Seed two milliliters of log phase growth insect SF9 cell culture evenly to the wells of a cell culture plate. Then allow the cells to attach for at least three hours at room temperature, in the hood.Using an inverted phase microscope at 250x, check the cell attachment. Then, remove the cell culture medium, and replace it with two milliliters of Grace's insect medium. After this, prepare transfection mixture A and B solutions according to the text protocol.Then gently mix transfection mixtures A and B together by tapping the tubes. Incubate the mixture at room temperature, in the hood, for 35 minutes. Next, add the mixture drop-wise, onto the seeded cells.Seal the wells with tape, and incubate the cells at 27 degrees Celsius, overnight. Replace the two milliliters of Grace's insect medium, with two milliliters of complete growth medium. Then, add 100 micromole Ganciclovir, to each well.Seal the wells with tape, and incubate the cells at 27 degrees Celsius for 72 hours. 72 hours post-infection, collect the medium from each well, and transfer it to 1.5 milliliter centrifuge tubes. Then centrifuge the tubes at 1500 times G, for five minutes at four degrees Celsius.Add one milliliter of complete growth medium to the well, and use a microscope with fluorescent light, to observe the infection signs of cells at the P1 baculovirus amplification stage. Transfer the supernatant into new 1.5 milliliter centrifuge tubes, and store them at four degrees Celsius, in dark conditions. After this, seed two milliliters of log phase growth insect SF9 cell culture, evenly, in the wells of a cell culture plate.Allow the cells to attach for at least three hours at room temperature, in the hood. Inoculate five microliters of P1 virus stock into the cell seeded wells, then, add 100 micromole Ganciclovir to each well. Seal the wells, and incubate the cells for 72 hours at 27 degrees Celsius.First, culture five milliliters of log phase growth insect SF9 cells, with complete growth medium, in T25 non-treated flasks. Then, add 25 microliters of P1 virus stock solution to infect the cell culture, for 48 hours, with gentle shaking, at 27 degrees Celsius. Using acetonitrile, prepare serial dilutions of 100 millimole permethrin.Then, seed 200 microliters of infected cell culture supplemented with 300 microliters of complete growth media, into a 24 well plate. Making sure that permethrin is fully dissolved in the acetonitrile, and evenly delivered into each well of the plate. After adding the permethrin, gently shake the plate to fully mix the permethrin in the medium.After this, seal the plate with tape, and incubate the plate at 27 degrees Celsius, for 48 hours, in dark conditions. Remove the cell culture medium from the plate without disturbing the bottom attached cell layer. Add 200 microliters of the MMT reagent to each well.Then, incubate the plate at 37 degrees Celsius for four hours, until dark purple formazan precipitants form in each well. Add 500 microliters of DMSO to each well, to fully dissolve the precipitants. Then transfer 200 microliters of the dissolved solution, to each well of a 96 well plate.Fully dissolving the precipitate into DMSO is crucial for the accuracy of the results. After adding the DMSO into samples in the plate, the samples will be washed using the pipette, for several times, until the precipitate has been fully dissolved. Finally, use a microplate reader to measure the absorbance values of the plate at 540 nanometers.At the P1 stage, the infection ratio is low. At the P2 stage, the infection ratio was significantly enhanced. At the P3 infection stage, almost all the cells showed symptoms of detachment from the cell culture plate, increases of cell diameter, as well as the cessation of cell growth.Based on the detected absorbance values, the Md alpha E7 expressing cells showed significantly higher viability, compared to the control CAT cells. While conducting this procedure, it is important to prevent cell contamination in all steps, by wiping the surface of stuffs, such as bottles and tubes, with 70 percentage ethanol before conducting the cell experiments. Following this procedure, some other methods, like the homology modeling and dark analysis, can be performed in order to answer additional questions, like the interactions between carboxyl esterase protein and permethrin ligand.This technique paved the way for the researchers in the insect toxicology, to explore the functional characterization of the genes and mechanisms in insecticide resistance of insects. Don't forget that working with insecticide can be very harmful, and precautions, such as gloves and lab coats, should always be taken while conducting these procedures.

Research

JoVE Journal

Biochemistry

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

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