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. udtryk og Isolation af Target proteiner med en Baculovirus-medieret insekt celle ekspressionssystem
<ol><li>Veje klon blunt-ended PCR produkter af target proteiner fra stuefluer.<ol>
<li>Design PCR primere af grøn fluorescerende proteiner (normal god landbrugspraksis) og hus flyve MdαE7 genet baseret på deres sekvenser og de særlige krav i den valgte vektor (tabel 1).</li>
		<li>Bruge en termostabil, korrekturlæsning DNA polymerase og primere fra trin 1.1.1 til at udføre en 150 µL PCR reaktio...

<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>

I de seneste årtier, har Heterolog ekspression systemer været udbredt at udtrykke og isolere store mængder proteiner, således at biokemiske og funktionelle beslutsomhed og karakterisering af enzymer i vitro. Til dato har flere forskellige modelsystemer, herunder Escherichia coli, Pichia pastoris, Sacccharomyces cerevisiaeog Spodoptera frugiperda er blevet tilpasset for rekombinante protein udtryk, og valget af de in vitro- system er afgørende for storstilet gener...

Insekticidresistens er i øjeblikket et stort problem på house flyve kontrol over hele verden1,2. Bestræbelser på at fastlægge mekanismen af insekticidresistens letter forståelsen af dette spørgsmål og dermed give nye strategier til at effektivt forebygge eller minimere spredning af resistens udvikling3. Carboxylesterases, har som en af de store afgiftning enzymer, tiltrukket en masse opmærksomhed for deres roller i binding og metaboliske insekticider i forskellige insekter4,5,6. Vores tidligere undersøgelse har identificeret flere carboxylesterases i stuefluer og deres udtryk niveauer var ikke kun constitutively op-reguleret i den resistent ALHF stamme, men også kan være induceret til højere niveauer i svar på permethrin behandlinger7 . De funktionelle karakterisering af disse carboxylesterase gener i metaboliske insekticider er dog stadig at blive udforsket.
Siden den første rapport i begyndelsen 1980s8, er en baculovirus-medieret fremmed gen expression system blevet bredt ansat på grund af sin høje protein produktionseffektivitet og eukaryote protein behandling kapaciteter9. Denne binære system består af to væsentlige elementer: den beregnede rekombinante baculovirus levere fremmede gener i værtsceller og storstilet udtryk for interesserede proteiner af celler inficeret af rekombinante baculovirus. I de seneste årtier, baculovirus medieret celle udtryk system har været meget anvendt til at producere tusindvis af rekombinante proteiner, lige fra cytosole enzymer til membran-bundet proteiner i insekter og pattedyr celler10. Vores tidligere undersøgelse har med held givet udtryk for flere CYP450 enzymer i insekt Sf9 celler med dette system11. I denne undersøgelse, vi konstrueret en carboxylesterase-rekombinant baculovirus for at inficere insekt Sf9 celler, udforskes celle tolerance over for forskellige permethrin behandlinger og storstilet udtrykt carboxylesterase proteiner i vitro for funktionelle udforskning. I stedet for at undersøge flere carboxylesterase isozyme blandinger fra insekt homogeniseret, som blev vedtaget af tidligere undersøgelser12,13, dette baculovirus-medieret insekt celle udtryk system tillader den specifikke udtryk og isolering af målrettede proteiner for bedre karakterisering af deres biokemiske og strukturelle egenskaber.
Tetrazolium salt-baseret analyse (MTT) er en høj overførselshastighed kolorimetriske metode udviklet og optimeret til måling af cellers levedygtighed. Denne analyse er baseret på den mekanisme, der kun levende celler er i stand til at metaboliske gul-farvet MTT reagens til en mørk lilla farvet formazankoncentrationen bundfald, som kan analyseres kolorimetrisk efter opløst i organiske opløsningsmidler14, 15. Flere mere præcise men tidskrævende metoder, såsom Trypan blå udstødelse og thymidine titrering assay16,17, er blevet udviklet i de seneste år. Dog er cellebaserede MTT assay stadig i øjeblikket anerkendes som den mest hurtig og let betjente metode til hurtigt opdage cellernes levedygtighed. Her bruger vi MTT analysen til at udforske den celle tolerance mod insekticidbehandling. Celler når inficeret med carboxylesterase rekombinante baculovirus stærkt forbedret tolerance understøtter carboxylesterases til insekticider, som til gengæld antyder deres inddragelse i insekticidresistens metaboliske roller.
Derudover blev en in vitro- metaboliske assay også gennemført i denne undersøgelse. Sammenlignet med almindelige carboxylesterase assays, der bruger fælles substrater såsom α-napthyl acetat (α-NA) og β-naphthyl acetat (β-NA) afspejler hydrolytisk aktiviteter af carboxylesterases, betragtes in vitro- metaboliske undersøgelse som en nøjagtig måde til direkte måling af carboxylesterases mod insekticider18aktiviteter. Denne metode har med held været ansat i forskellige insekter til at karakterisere flere cytokrom P450s i forening med insekticid resistens11,19,20. Dog, denne metode har ikke endnu blevet anvendt i carboxylesterase undersøgelser. Med tilgængeligheden af carboxylesterase proteiner produceret af baculovirus-medieret ekspressionssystem, kan vi udføre en in vitro- metaboliske undersøgelse af carboxylesterases mod permethrin, som yderligere kan give stærke beviser for deltagelse af carboxylesterases i tildeling pyrethroid modstand i hus fluer.

<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-medieret stofskifte menes at spille en større rolle i insekticidresistens i forskellige insekter. Flere carboxylesterase gener blev fundet op-reguleret i resistent hus flyve stamme, der henviser til, at deres roller i tildeling insekticidresistens forblev for at blive udforsket. Her, vi har udviklet en protokol for den funktionelle karakterisering af carboxylesterases. Tre eksempel eksperimenter præsenteres: (1) udtryk og isolering af carboxylesterase proteiner gennem en baculovirus-medieret insekt Spodoptera frugiperda (Sf9) celle ekspressionssystem; (2) en celle-baserede MTT (3-[4, 5-dimethykthiazol-2-yl] -2, 5-diphenyltetrazolium bromid) cytotoksicitet assay at måle tolerance over for insekt celler til forskellige permethrin behandlinger; og (3) in vitro- metaboliske undersøgelser for at udforske de metaboliske funktioner i carboxylesterases mod permethrin. Carboxylesterase gen MdαE7 var klonet fra en resistent hus flyve stamme ALHF og bruges til at konstruere en rekombinante baculovirus for Sf9 celler infektion. Celle viabilities mod forskellige permethrin behandlinger blev målt med MTT-analysen. Den forbedrede celle tolerance af den eksperimentelle gruppe (MdαE7-rekombinant baculovirus inficerede celler) sammenlignet med kontrolgruppen (kat-rekombinant baculovirus inficerede celler og normal god landbrugspraksis-rekombinant baculovirus inficerede celler) at permethrin behandlinger foreslået funktionerne i MdαE7 i metaboliske insekticider, dermed beskytte celler mod kemiske skader. Udover det, var carboxylesterase proteiner udtrykt i insekt celler, Sf9 og isoleret for at foretage en in vitro- metaboliske undersøgelse. Vores resultater viste en betydelig in vitro- metaboliske effektivitet af MdαE7 mod permethrin, direkte antyder inddragelse af carboxylesterases i metaboliske insekticider og dermed overdrage insekticidresistens i hus fluer.

Cellernes levedygtighed mod forskellige permethrin behandlinger (MTT assay)
Cytotoksicitet af permethrin blev undersøgt i MdαE7-rekombinant baculovirus inficerede Sf9 celler (eksperimentelle gruppe) og kat-rekombinant baculovirus (leveres af baculovirus inficeret kit) inficerede celler (kontrolgruppen). De forbedrede celle tolerancer til permethrin i MdαE7 udtrykker celler kraftigt støtte de metaboliske roller af denne carboxyl...

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

Vi præsenterer her, en protokol for at producere hus flyve carboxylesterase proteiner i vitro med en baculovirus medieret insekt celle ekspressionssystem og senere funktionelt karakteriserer deres roller i metaboliske permethrin, dermed overdrage pyrethroid modstand af celle-baseret MTT assay og in vitro- metaboliske undersøgelser.

Funktionelle karakterisering af Carboxylesterases i insekticid resistente stuefluer, 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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