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. expresión y aislamiento de proteínas de la blanco con un sistema de expresión celular de insecto Baculovirus-mediada
<ol><li>Direccionalmente clon punta Roma los productos PCR de las proteínas blanco de moscas domésticas.<ol>
<li>Diseño de cartillas de la polimerización en cadena de la proteína verde fluorescente (GFP) y el gen de MdαE7 mosca de la casa basado en sus secuencias y las necesidades especiales del vector elegido (tabla 1).</li>
		<li>Utilice una ADN polimerasa termoestable, cor...

<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>

En las últimas décadas, sistemas de expresión heteróloga se han utilizado ampliamente para expresar y para aislar grandes cantidades de proteínas, permitiendo la determinación bioquímica y funcional y caracterización de enzimas en vitro. Hasta la fecha, varios sistemas de modelo diferente, incluyendo Escherichia coli, Sacccharomyces cerevisiae, Pichia pastorisy Spodoptera frugiperda han sido adaptados para la expresión de la proteína recombinante y la elección de la...

Resistencia a los insecticidas es actualmente un grave problema de casa control de moscas en todo el mundo1,2. Esfuerzos para determinar el mecanismo de resistencia a los insecticidas facilita la mejor comprensión de este tema y así proporcionar nuevas estrategias para efectivamente prevenir o minimizar la propagación de resistencia desarrollo3. Carboxilesterasas, como una de las enzimas de desintoxicación más importantes, han atraído mucho la atención por su participación en el secuestro y metabolizan insecticidas en varios insectos4,5,6. Nuestro estudio anterior ha identificado múltiples carboxilesterasas en casa vuelan y sus niveles de expresión no sólo fueron constitutivamente para arriba-regulados en la cepa ALHF resistente pero también pueden ser inducido a mayores niveles en respuesta a los tratamientos de permetrina7 . Sin embargo, la caracterización funcional de estos genes carboxilesterasa en metabolizan insecticidas aún explorarse.
Desde el primer informe de principios de la década de 19808, un sistema de expresión de baculovirus-mediada gen extranjero ha sido empleado ampliamente debido a su eficiencia de producción de alto valor proteico y proteína eucariota procesamiento capacidades9. Este sistema binario se compone de dos elementos esenciales: el baculovirus recombinante construcción entrega de genes foráneos en las células del huésped y la expresión a gran escala de proteínas interesadas por las células infectadas por el baculovirus recombinante. En las últimas décadas, el sistema de expresión de baculovirus mediada por células ha sido ampliamente utilizado para producir miles de proteínas recombinantes, que van desde enzimas citosólicas a las proteínas de membrana-limitan en insectos y mamíferos las células10. Nuestro estudio anterior ha expresado con éxito múltiples enzimas CYP450 en insectos células Sf9 con este sistema11. En este estudio, construido un baculovirus recombinante carboxilesterasa para infectar insectos células Sf9, exploró la tolerancia celular a permetrina diferentes tratamientos y a gran escala carboxilesterasa expresa proteínas en vitro para funcional exploración. En lugar de investigar múltiples mezclas de isoenzima carboxilesterasa de insectos homogenados adoptados por anteriores estudios12,13, este sistema de expresión celular de insecto baculovirus-mediada permite la expresión concreta y aislamiento de proteínas específicas para la mejor caracterización de sus propiedades bioquímicas y estructurales.
El ensayo basado en la sal de tetrazolio (MTT) es un método colorimétrico de alto rendimiento desarrollado y optimizado para medir la viabilidad de las células. Este ensayo se basa en el mecanismo que sólo las células vivas son capaces de metabolizar el reactivo MTT de color amarillo a un precipitado precipitado de color púrpura oscuro, que puede ser analizado por después de disuelto en solventes orgánicos14, 15. Varios más preciso pero requiere mucho tiempo métodos, como la exclusión azul de tripano y la timidina titulación ensayo16,17, se han desarrollado en los últimos años. Sin embargo, el ensayo MTT celular aún en la actualidad es reconocido como el método más rápido y fácil manejo para detectar rápidamente la viabilidad celular. Aquí, utilizamos el ensayo MTT para explorar la tolerancia de la célula contra los tratamientos insecticidas. La mayor tolerancia de las células cuando infectadas con baculovirus recombinante carboxilesterasa fuertemente apoya las funciones metabólicas de carboxilesterasas a los insecticidas, que a su vez, sugiere su participación en la resistencia a los insecticidas.
Además, un ensayo en vitro metabólica también se realizó en este estudio. En comparación con ensayos de carboxilesterasa general que utilizan sustratos comunes tales como acetato de α-napthyl (α-NA) y β-naftil acetato (β-NA) para reflejar actividades hidrolíticas de carboxilesterasas, estudio metabólico en vitro es considerado como una forma precisa medir directamente actividades de carboxilesterasas hacia insecticidas18. Este método se ha empleado con éxito en varios insectos para caracterizar varios cytochrome P450s en asociación con insecticida resistencia11,19,20. Sin embargo, este método ha no aún ha aplicado en estudios de la carboxilesterasa. Con la disponibilidad de la carboxilesterasa proteínas producidas por el sistema de expresión de baculovirus-mediada, podemos realizar un estudio metabólico en vitro de carboxilesterasas hacia permetrina, que puede proporcionar más fuerte evidencia de la participación de carboxilesterasas en confiriendo resistencia a piretroides en casa vuela.

<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

Metabolismo mediado por la carboxilesterasa se piensa para desempeñar un papel importante en la resistencia a los insecticidas en los insectos varios. Varios genes de la carboxilesterasa fueron encontrados para arriba-regulados en la cepa mosca de casa resistente, mientras que seguía habiendo sus papeles en que confieren resistencia a los insecticidas para ser explorado. Aquí, hemos diseñado un protocolo para la caracterización funcional de carboxilesterasas. Se presentan tres experiencias de ejemplo: (1) expresión y aislamiento de proteínas de la carboxilesterasa a través de un insecto mediada por baculovirus Spodoptera frugiperda (Sf9) sistema de expresión celular; (2) torneo de mesas múltiples basadas en células (3-[4, 5-dimethykthiazol-2-yl] -2, 5-diphenyltetrazolium bromuro) ensayo de citotoxicidad para medir la tolerancia de las células de insecto permethrin diferentes tratamientos; y (3) en vitro estudios metabólicos para explorar las capacidades metabólicas de carboxilesterasas hacia permetrina. El gen de la carboxilesterasa MdαE7 fue clonado de una casa resistente a mosca cepa ALHF y utilizados para construir un baculovirus recombinante para la infección de las células Sf9. Las viabilidades de célula contra permethrin diferentes tratamientos fueron medidos en el ensayo MTT. La tolerancia de la célula mejorada del grupo experimental (células de MdαE7 recombinante baculovirus infectadas) comparado con los de los grupos de control (CAT-recombinante baculovirus infectadas células y GFP-recombinante baculovirus infectadas células) a la permetrina tratamientos sugieren las capacidades de MdαE7 en metabolizan insecticidas, protegiendo las células contra los daños químicos. Además, carboxilesterasa proteínas fueron expresadas en células Sf9 insectos y aisladas para llevar a cabo un estudio metabólico en vitro . Nuestros resultados indican una significativo en la vitro eficiencia metabólica de MdαE7 hacia la permetrina, indicando directamente la participación de carboxilesterasas en metabolizan insecticidas y así que confieren resistencia a los insecticidas en casa vuela.

La viabilidad de las células hacia la permetrina diferentes tratamientos (ensayo MTT)
La citotoxicidad de la permetrina fue examinada en las células MdαE7-recombinante baculovirus infectadas Sf9 (grupo experimental) y CAT-recombinante baculovirus (proporcionado por el kit de baculovirus infectadas) infectado células (grupos control). Las tolerancias de célula mejorada a la permetrina en MdαE7 expresando fuertemente las célul...

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

Aquí, presentamos un protocolo para producir casa mosca carboxilesterasa proteínas en vitro con un sistema de expresión de baculovirus mediada por células de insectos y caracterizar funcionalmente más adelante su papel en la metabolización de permetrina, de tal modo, que confieren piretroides resistencia mediante la realización de celular MTT análisis y en vitro estudios metabólicos.

Caracterización funcional de carboxilesterasas en casa resistente al insecticida moscas, 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

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

Caracterización estructural de polisacáridos de pared celular mananos en plantas con ritmo

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts ...

Investigación

Bioquímica

Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

Protocolo de mejora para la inmunoprecipitación de cromatina de músculo esquelético de ratón

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a ...

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Caracterización de las glicoproteínas con el doblez de la inmunoglobulina por cristalografía de rayos x y técnicas biofísicas

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

Fluorescent Silver Staining of Proteins in Polyacrylamide Gels

Tinción fluorescente de plata de proteínas en geles de poliacrilamida

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide ...

Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis

Caracterización basada en fluorescencia rápida de solo vesículas extracelulares en la sangre humana con el análisis de seguimiento de nanopartículas

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released ...

Identification of Functional Protein Regions Through Chimeric Protein Construction

Identificación de regiones de la proteína funcional a través de la construcción de la proteína quimérica

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Caracterización de transportadores de membranas por expresión heteróloga en E. coli y producción de vesículas de membrana

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...

SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

SUMO-Binding Entities SUBEs as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Entidades de unión SUMO (SUBE) como herramientas para el enriquecimiento, aislamiento, identificación y caracterización del proteome SUMO en cáncer de hígado

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in ...

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Caracterización funcional de las ligasinas de ubiquitina RING-Tipo E3 In Vitro y In Planta

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent ...

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Caracterización de estructuras amiloideas en el envejecimiento C. Elegans usando imágenes de por vida de fluorescencia

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These ...