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. ifade ve yalıtım bir böcek hücre Baculovirus-aracılı ifade sistemi ile hedef proteinlerin
<ol><li>Directionally evi sinekler üzerinden hedef proteinlerin PCR ürünleri künt uçlu klon.<ol>
<li>PCR astar yeşil flüoresan protein (GFP) ve ev sinek MdαE7 gene onların dizileri ve seçtikleri vektörü (Tablo 1) özel gereksinimleri temel tasarım.</li>
		<li>Bir transfer, prova-okuma DNA polimeraz ve astar adım 1.1.1 150 µL PCR reaksiyon (30 µL tepki arabelleği, 10 mM dNTPs, DNA polimera...

<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. 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Son yıllarda, kapaklı ifade sistemleri yaygın olarak hızlı ve büyük miktarda protein, böylece biyokimyasal ve fonksiyonel belirlenmesi ve enzimler vitrokarakterizasyonu izin izole etmek için kullanılmıştır. Bugüne kadar Escherichia coli, Pichia pastoris, Sacccharomyces cerevisiaeve Spodoptera frugiperda gibi birkaç farklı model sistemleri Rekombinant protein ifade ve seçim için adapte edilmiştir vitro sistem baktılar proteinler22<...

Böcek ilacı direnç Şu anda evi sinek denetim dünya çapında1,2önemli bir konudur. Böcek ilacı direnç mekanizmasının belirlemek için çabaları bu konunun daha iyi anlamayı kolaylaştırır ve böylece etkili önlemek veya direnç geliştirme3yayılmasını en aza indirmek için roman stratejileri sağlamak. Carboxylesterases, bir büyük detoksifikasyon enzimler olarak iyon ve böcek öldürücüler çeşitli böcekler4,5,6metabolize rolleri için ilgi çok çekmiştir. Bizim önceki çalışma evi sinekler içinde birden çok carboxylesterases tespit etti ve onların ifade düzeyleri sadece yapısal dayanıklı ALHF zorlanma yukarı düzenlenir değildi ama aynı zamanda yanıt olarak Permetrin tedaviler7 indüklenen için daha yüksek düzeyde olabilir . Ancak, böcek ilaçları metabolize içinde bu carboxylesterase gen işlevsel karakterizasyonu keşfedilmeyi kalır.
Erken 1980'li yıllarda8ilk raporda beri yabancı gen baculovirus-aracılı ifade sistem yaygın olarak yüksek protein üretim verimliliği ve ökaryotik protein yetenekleri9işleme nedeniyle istihdam edilmiştir. Bu ikili sistem iki temel unsurdan oluşur: konak hücreleri ve büyük ölçekli ifade rekombinant baculovirus tarafından enfekte hücreleri tarafından baktılar proteinlerin içine yabancı gen teslim inşa rekombinant baculovirus. Son on yıl boyunca baculovirus aracılı hücre ifade sistem yaygın rekombinant proteinler membran bağlı proteinler arasında böcek sitozolik enzimler arasında değişen, binlerce üretmek için kullanılmıştır ve10memeli hücreleri. Bizim önceki çalışma başarıyla birden fazla CYP450 enzimleri bu sistem11ile böcek Sf9 hücrelerdeki dile getirdi. Bu çalışmada, böcek Sf9 hücre bulaştırmak için bir carboxylesterase-rekombinant baculovirus inşa, farklı Permetrin tedaviler ve büyük ölçekli ifade carboxylesterase proteinler vitro için hücre tolerans incelenmiştir fonksiyonel keşif. Önceki çalışmalar12,13tarafından kabul edilen birden çok carboxylesterase izozim karışımları böcek homogenates üzerinden araştıran yerine, bu böcek hücre baculovirus-aracılı ifade sistemi belirli ifade sağlar ve biyokimyasal ve yapısal özelliklerini daha iyi karakterizasyonu için hedeflenen proteinlerin yalıtım.
Tetrazolium tuz tabanlı tahlil (MTT) geliştirilen ve hücre canlılığı ölçmek için optimize edilmiş yüksek üretilen iş kolorimetrik yöntemi olduğunu. Bu tahlil sadece canlı hücreler sarı renkli MTT reaktif colorimetrically sonra çözünmüş organik çözücüler14'analiz edilebilir bir koyu mor renkli formazan çökelti için metabolize yeteneğine mekanizması dayanır, 15. Birkaç daha doğru ama Trypan mavi dışlama ve timidin titrasyon tahlil16,17, gibi zaman alan yöntemleri son yıllarda geliştirilmiştir. Ancak, hücre tabanlı MTT tahlil hala şu anda hızlı bir şekilde hücre canlılığı algılamak için en hızlı ve kolay işletilen yöntemi olarak tanınır. Burada, böcek ilacı tedaviler karşı hücre tolerans keşfetmek için MTT tahlil kullanırız. Güçlü carboxylesterase rekombinant baculovirus ile enfekte hücreleri geliştirilmiş dayanıklılık için böcek öldürücüler, buna karşılık onların katılımı böcek ilacı direnç www.cdc.gov/drugresistance öneriyor carboxylesterases metabolik rolleri destekleyen.
Ayrıca, bir vitro metabolik tahlil de bu çalışma yapılmıştır. Carboxylesterases hydrolytic faaliyetleri yansıtmak için α-napthyl asetat (α-NA) ve β-naftil asetat (β-NA) gibi ortak yüzeylerde kullanın genel carboxylesterase deneyleri ile karşılaştırıldığında, vitro metabolik çalışma doğru bir yol kabul edilmektedir doğrudan carboxylesterases böcek öldürücüler18doğru faaliyetlerinin ölçmek için. Bu yöntem başarılı bir şekilde çeşitli böcek böcek ilacı direnç11,19,20ile birlikte birden çok sitokrom P450s karakterize etmek için istihdam edilmiştir. Ancak, bu yöntem henüz carboxylesterase çalışmalarda uygulanmamıştır. Baculovirus-aracılı ifade sistem tarafından üretilen carboxylesterase proteinler durumu ile daha da güçlü kanıtı sağlayabilir Permetrin doğru carboxylesterases bir vitro metabolik çalışma gerçekleştirebilirsiniz pyrethroid direnç içinde ev veriyor, carboxylesterases, uçar.

<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-aracılı metabolizma çeşitli böcek böcek ilacı direnci önemli bir rol oynamaktadır düşünülmektedir. Böcek ilacı direnci veriyor onların rolleri keşfedilmeyi kaldı, ancak birkaç carboxylesterase genler dayanıklı ev sinek gerginliği, yukarı düzenlenir bulunamadı. Burada, biz bir protokol carboxylesterases fonksiyonel karakterizasyonu için tasarlanmıştır. Üç örnek deneyler sunulmaktadır: (1) ifade ve yalıtım carboxylesterase protein aracılığıyla bir baculovirus-aracılı böcek Spodoptera frugiperda (Sf9) hücre ifade sistemi; (2) bir hücre tabanlı MTT (3-[4, 5-dimethykthiazol-2-yl] -2, 5-diphenyltetrazolium bromür) böcek hücreleri toleransları farklı Permetrin tedavilere; ölçmek için sitotoksisite tahlil ve carboxylesterases doğru Permetrin metabolik yeteneklerini keşfetmek için (3) vitro metabolik çalışmaları. MdαE7 dayanıklı bir evden klonlanmış carboxylesterase gen soy ALHF sinek ve Sf9 hücreleri enfeksiyon için rekombinant baculovirus oluşturmak için kullanılan. Hücre viabilities farklı Permetrin tedaviler karşı MTT tahlil ile ölçüldü. Permetrin için denetim gruplarının (kedi-rekombinant baculovirus enfekte hücreleri ve GFP-rekombinant baculovirus enfekte hücreleri) ile karşılaştırıldığında deney grubu (MdαE7-rekombinant baculovirus enfekte hücreleri) Gelişmiş hücre toleransları tedaviler MdαE7 yeteneklerini böylece hücrelerin kimyasal zararlardan korunması, böcek öldürücüler, metabolize önerdi. Bunun yanı sıra, carboxylesterase proteinler böcek Sf9 hücrelerde ifade ve vitro metabolik çalışma yapmak için izole. Bizim sonuçları önemli vitro bir metabolik verimlilik MdαE7 Permetrin, doğrudan böcek ilaçları metabolize içinde carboxylesterases tutulumu gösteren doğru belirtilen ve böylece evde böcek ilacı direnci veriyor uçar.

Hücre canlılığı farklı Permetrin tedaviler (ÇMT tahlil) doğru
Permetrin sitotoksisite MdαE7-rekombinant enfekte baculovirus Sf9 hücreleri (deney grubu) ve kedi-rekombinant (enfekte baculovirus kit tarafından sağlanan) baculovirus enfekte hücreleri (kontrol grubu) incelenmiştir. Gelişmiş hücre toleransları için hücreleri kuvvetle ifade Permetrin MdαE7 içinde bu carboxylesterase böcek öldürücüler ve böylec...

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

Burada, bir aracılı baculovirus böcek hücre ifade sistemi ile ev sinek carboxylesterase proteinler vitro üretmek ve daha sonra işlevsel olarak Permetrin, metabolize onların rolleri böylece, karakterize bir protokol pyrethroid veriyor mevcut rezistansının hücre tabanlı MTT tahlil ve vitro metabolik çalışmalar yürütmektedir.

Carboxylesterases böcek ilacı dayanıklı ev sinekleri, sinek Domestica içinde işlevsel karakterizasyonu

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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