Name: transient expression of foreign genes in insect cells (sf9) for protein functional assay
Uploaded: 2018-02-22T13:00:00
Description: Watch this Scientific Journal Video about Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay at JoVE.com

Agradecemos a Dr. Jian Horng Leu del Instituto de Biología Marina, Universidad Nacional de mar de Taiwán para proporcionar 3 construcciones de plásmido. Esta investigación fue apoyada por Grant 106-2311-B-197-001 - desde el Ministerio de ciencia y tecnología (MOST).

1. preparaciones
<ol><li>Cultivo de células de insecto<ol>
<li>Preparar 50 mL de medio de cultivo celular. Para ello, Añadir 500 μl de antibióticos (anfotericina B = 0,25 μg/mL, penicilina = 100 unidades/mL, estreptomicina 100 μg/ml) y 5 mL de suero bovino fetal inactivado con calor en medio de cultivo celular sin suero (sin FBS o antibióticos). Nota: Calentar el suero fetal bovino a 65 ° C por 30 min en un baño de agua antes de su uso.</li>
		<li>Mantener Spodoptera frugiperda (Le...

<ol>
	<li>Miller, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculoviruses+as+gene+expression+vectors.">Baculoviruses as gene expression vectors.</a> Annual Reviews in Microbiology. 42 (1), 177-199 (1988).</li><li>Theilmann, D. A., Fauquet, C. M., Mayo, M. A., Maniloff, J., Desselberger, U., Ball, L. A., 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=Family+Baculoviridae.">Family Baculoviridae.</a> Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses. , 1129-1185 (2005).</li><li>Nai, Y. S., Huang, Y. F., Chen, T. H., Chiu, K. P., Wang, C. H., Shields, V. D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+nucleopolyhedrovirus'+taxonomic+position.">Determination of nucleopolyhedrovirus' taxonomic position.</a> Biological Control of Pest and Vector Insects. , 169-200 (2017).</li><li>Friesen, P. D., Miller, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+baculovirus+early+gene+expression.">Regulation of baculovirus early gene expression.</a> The Baculoviruses. , 141-170 (1997).</li><li>Smith, G. E., Summers, M., Fraser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+human+beta+interferon+in+insect+cells+infected+with+a+baculovirus+expression+vector.">Production of human beta interferon in insect cells infected with a baculovirus expression vector.</a> Mol. Cell. Biol. 3 (12), 2156-2165 (1983).</li><li>Luckow, V. A., Summers, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+the+development+of+baculovirus+expression+vectors.">Trends in the development of baculovirus expression vectors.</a> Nature Biotechnol. 6 (1), 47-55 (1988).</li><li>Jarvis, D. L., Finn, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modifying+the+insect+cell+N-glycosylation+pathway+with+immediate+early+baculovirus+expression+vectors.">Modifying the insect cell N-glycosylation pathway with immediate early baculovirus expression vectors.</a> Nature Biotechnol. 14 (1996), 1288-1292 (1996).</li><li>Clem, R. J., Miller, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+programmed+cell+death+by+the+baculovirus+genes+p35+and+iap.">Control of programmed cell death by the baculovirus genes p35 and iap.</a> Mol. Cell. Biol. 14, 5212-5222 (1994).</li><li>Leu, J. H., Kuo, Y. C., Kou, G. H., Lo, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+and+characterization+of+an+inhibitor+of+apoptosis+protein+(IAP)+from+the+tiger+shrimp,+Penaeus+monodon.">Molecular cloning and characterization of an inhibitor of apoptosis protein (IAP) from the tiger shrimp, Penaeus monodon.</a> Dev. Comp. Immunol. 32, 121-133 (2008).</li><li>Zhao, Y. G., Eggleston, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+promoters+for+transient+gene+expression+in+cultured+mosquito+cells.">Comparative analysis of promoters for transient gene expression in cultured mosquito cells.</a> Insect Mol. Biol. 8 (1), 31-38 (1999).</li><li>Nai, Y. S., Yang, Y. T., Kim, J. S., Wu, C. Y., Chen, Y. W., Wang, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculoviral+IAP2+and+IAP3+encoded+by+Lymantria+xylina+multiple+nucleopolyhedrovirus+(LyxyMNPV)+suppress+insect+cell+apoptosis+in+a+transient+expression+assay.">Baculoviral IAP2 and IAP3 encoded by Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV) suppress insect cell apoptosis in a transient expression assay.</a> Appl. Entomol. Zool. 51, 305-316 (2016).</li><li>Eslami, A., Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+Blotting:+Sample+Preparation+to+Detection.">Western Blotting: Sample Preparation to Detection.</a> J. Vis. Exp. (44), e2359 (2010).</li><li>. Basic Methods in Cellular and Molecular Biology. Separating Protein with SDS-PAGE Available from: <a target="_blank" href="https://www.jove.com/science-education/5058/separating-protein-with-sds-page">https://www.jove.com/science-education/5058/separating-protein-with-sds-page</a> (2017)</li><li>Vucic, D., Kaiser, W. J., Harvey, A. J., Miller, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+reaper-induced+apoptosis+by+interaction+with+inhibitor+of+apoptosis+proteins+(IAPs).">Inhibition of reaper-induced apoptosis by interaction with inhibitor of apoptosis proteins (IAPs).</a> Proc. Natl. Acad. Sci. USA. 94, 10183-10188 (1997).</li></ol>

El concepto de calor basado en el choque pDHsp/V5-su/sf9 celular sistema primero fue descrito por Clem et en 19948. Comparación del promotor del gen baculovirales (IE1) y hsp70 de Drosophila demostraron que hsp70 tenía una eficacia más alta en células de mosquito10. Además, debido a la inducción del choque de calor, el tiempo de expresión de la proteína podría ser controlado precisamente después del tratamiento de choque de calo...

Dos sistemas de expresión de proteínas se han utilizado comúnmente para la producción de proteínas: sistemas de expresión de proteínas de célula procariota (sistema de expresión de genes deEscherichia coli ) y sistemas de expresión de proteínas eucariotas. Un sistema de expresión de proteínas eucariotas popular es la expresión del baculovirus vector sistema (BEVS)1. Primero se utilizaron los baculovirus como agentes de control biológico en todo el mundo de la agricultura y las plagas del bosque. En las últimas décadas, baculovirus fueron desarrollados como herramientas biotecnológicas para vectores de la expresión de la proteína así. Los genomas de baculovirus consisten en ADN circular de doble hebra y envuelta nucleocápside2. Hasta la fecha, más de setenta y ocho baculovirus aislados han sido secuenciados3. Basado en la cascada de temporal expresiones baculovirales gene en las células del insecto huésped, la transcripción del gen podría clasificarse en cuatro cascadas temporales, incluyendo inmediata temprana, genes retrasado-temprano, tarde y muy tarde4.
BEVSs fueron señalados para que los promotores de genes muy tarde (es decir, poliedro o p10 promotor) fueron utilizados para conducir los genes de la blanco, mientras que el baculovirus recombinante fue generada por recombinación homóloga. Expresión de proteínas extrañas en las células de los insectos por baculovirus recombinante es similar a la de proteínas de mamíferas en modificaciones post-traduccionales (adecuadas para la producción de glicoproteínas). Así, el baculovirus ha sido ampliamente utilizado5,6,7. Sin embargo, una limitación es la presencia de diferentes vías de N-glicosilación en células de insecto7.
Por lo tanto, se desarrolló un nuevo sistema de expresión de baculovirus, el sistema de expresión transitoria del gen. Este sistema expresa genes extranjeros en la unidad de promotores inmediata temprana baculovirales (promotor deie-1 ) en células de los insectos. Mediante este sistema, la proteína diana puede expresarse inmediatamente bajo el control del promotor es decir-1 mientras modifica el camino de N-glicosilación en células de los insectos, resultando en mejor oligosacáridos N-ligados7. Por otra parte, genes inmediatos tempranos baculovirales están transcritos por la polimerasa II del RNA de la célula huésped y no requieren ningún factor viral para activación4. Por lo tanto, las proteínas extranjeras pueden expresarse en células de los insectos dentro de poco tiempo. Hasta la fecha, el transitorio sistema de expresión del gene es una de las más importantes tecnologías para realizar análisis funcional de la proteína en el sistema de cultivo de baculovirus en vitro de la célula. El sistema se puede aplicar para analizar la función de baculovirus o proteínas extrañas. Uno de los sistemas de expresión génica transitoria disponible comercialmente se basa en los promotores de genes inmediatos tempranos (IE) de Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) (OpIE2 y OpIE1 promotores).
Sin embargo, el menor nivel de expresión de genes foráneos en células de los insectos es todavía un problema cuando el sistema de expresión de gen transitoria basada en promotor de OpIE fue usado8,9,10. Por lo tanto, otro sistema de expresión transitoria del gen fue construido basado en el promotor de la Drosophila calor choque proteína 70 (hsp70) gen8,9. El promotor de la hsp70 funciona más eficientemente que el promotor baculovirales IE cuando inducida por choque del calor en células de los insectos10. En este sistema, los genes de la blanco se expresaron bajo la impulsión de 70 (Dhsp70) promotor de Drosophila calor choque. Genes foráneos pueden clonarse fácilmente en el plásmido de expresión génica transitoria por métodos de clonación basados en PCR. Además, el control de la sincronización para expresión génica puede realizarse por la inducción del choque de calor.
En este informe, seguir el enfoque y expresar tres diferentes truncamientos del gen baculovirales (inhibidor de la apoptosis 3, iap3 de Monacha xylina MNPV) mediante el sistema de expresión de proteína de choque transitorio de calor y más se aplican estas proteínas expresadas en el análisis de la actividad anti-apoptótica. Este sistema tampoco puede expresar las proteínas extranjeras rápidamente o ser más aplicado a la evaluación de la actividad de la proteína anti-apoptótica en las células sf9, teniendo también la posibilidad de ser aplicado a otros ensayos de actividad de la proteína.

<table>
	<tbody>
		<tr>
			<td>Antibiotic-Antimycotic, 100X</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td>for insect cell culture</td>
		</tr>
		<tr>
			<td>Certified Foetal Bovine Serum</td>
			<td>Bioind</td>
			<td>04-001-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sf-900 II SFM</td>
			<td>Thermo Fisher</td>
			<td>10902096</td>
			<td>serum-free cell culture medium</td>
		</tr>
		<tr>
			<td>Sf9 cells</td>
			<td>ATCC</td>
			<td>CRL-1711</td>
			<td></td>
		</tr>
		<tr>
			<td>25cm2 cell culture flask</td>
			<td>Nunc, Thermo Fisher</td>
			<td>156340</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted light microscopy</td>
			<td>WHITED</td>
			<td>WHITED WI-400</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC HIT Competent Cell</td>
			<td>Bioman</td>
			<td>RH618-J80</td>
			<td>Escherichia coli (DH5&#945;)</td>
		</tr>
		<tr>
			<td>L.B. Broth (Miller)</td>
			<td>Bioman</td>
			<td>LBL407</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar, Bacteriological Grade</td>
			<td>Bioman</td>
			<td>AGR001</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeocin</td>
			<td>Invitrogen</td>
			<td>ant-zn-1</td>
			<td>selection antibiotic</td>
		</tr>
		<tr>
			<td>PCR Master Mix (2X)</td>
			<td>ThermoFisher</td>
			<td>K0171</td>
			<td></td>
		</tr>
		<tr>
			<td>Geneaid Midi Plasmid Kit (Endotoxin Free)</td>
			<td>Geneaid</td>
			<td>PIE25</td>
			<td></td>
		</tr>
		<tr>
			<td>Actinomycin D</td>
			<td>SIGMA</td>
			<td>A9415</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 50 mL centrifuge tubes</td>
			<td>SIGMA</td>
			<td>CLS430829-500EA</td>
			<td>50 mL tubes</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Gizmo Supply Co</td>
			<td>B-CNT-SLDE-V2</td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well Multidish</td>
			<td>Nunc, Thermo Fisher</td>
			<td>142475</td>
			<td>24-well plate</td>
		</tr>
		<tr>
			<td>Cellfectin II Reagent</td>
			<td>Thermo Fisher</td>
			<td>10362100</td>
			<td>cell transfectin reagent</td>
		</tr>
		<tr>
			<td>PBS-Phosphate-Buffered Saline (10X) pH 7.4</td>
			<td>Thermo Fisher</td>
			<td>AM9624</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#215;SDS Loading Dye</td>
			<td>Bioman</td>
			<td>P1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilon-P (PVDF Blotting Membranes)</td>
			<td>Merck Milipore</td>
			<td>IPVH00010</td>
			<td>PVDF membranes</td>
		</tr>
		<tr>
			<td>Mini Trans-Blot Cell system</td>
			<td>BIO-RED</td>
			<td>1703930</td>
			<td>Blotting device</td>
		</tr>
		<tr>
			<td>Ponceau S solution</td>
			<td>SIGMA</td>
			<td>6226-79-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-V5</td>
			<td>SIGMA</td>
			<td>V8137</td>
			<td>rabbit anti-V5 antibody</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG-horseradish peroxidase (HRP)</td>
			<td>Jackson</td>
			<td>111-035-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Merck</td>
			<td>817072</td>
			<td></td>
		</tr>
		<tr>
			<td>6-Well Multidish</td>
			<td>Nunc, Thermo Fisher</td>
			<td>145380</td>
			<td></td>
		</tr>
		<tr>
			<td>0.4 % trypan blue solution</td>
			<td>AMRESCO</td>
			<td>K940-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>P10 pipetman</td>
			<td>Gilson</td>
			<td>F144802</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 pipetman</td>
			<td>Gilson</td>
			<td>F123602</td>
			<td></td>
		</tr>
		<tr>
			<td>Tape</td>
			<td>Symbio</td>
			<td>PPS7</td>
			<td>24 well tape ( 19 mm&#215;36 M)</td>
		</tr>
	</tbody>
</table>

transient expression of foreign genes in insect cells (sf9) for protein functional assay

El sistema de expresión transitoria del gen es una de las tecnologías más importantes para la realización de análisis funcionales de la proteína en el sistema de cultivo de baculovirus en vitro de la célula. Este sistema fue desarrollado para extranjeros expresan genes bajo el control del promotor baculovirales en plásmidos de expresión transitoria. Además, este sistema puede aplicarse a un análisis funcional de proteínas extrañas o baculovirus sí mismo. El sistema de expresión transitoria del gen más ampliamente y comercialmente disponible se desarrolla basado en el promotor de genes inmediatos tempranos (IE) de Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). Sin embargo, se observó un nivel de baja expresión de genes foráneos en células de los insectos. Por lo tanto, un sistema de expresión transitoria del gen fue construido para mejorar la expresión de la proteína. En este sistema, plásmidos recombinantes fueron construidos para contener la secuencia de destino bajo el control del Drosophila calor choque 70 (Dhsp70) promotor. Este protocolo presenta la aplicación de este calor basado en el choque pDHsp/V5-su (epitope V5 con 6 histidina)Spodoptera frugiperda Handy sistema (células sf9); Este sistema está disponible no sólo para la expresión de genes sino también para la evaluación de la actividad de anti-apoptotic de candidato proteínas en células de los insectos. Además, este sistema puede ser bien transfectado con un plásmido recombinante o transfected Co dos plásmidos recombinantes potencialmente funcionalmente antagónicos en células de los insectos. El protocolo demuestra la eficacia de este sistema y proporciona un caso práctico de esta técnica.

Toda la longitud y otros dos truncamientos (dominios BIR y anillo) de Ly-IAP3 de LyxyMNPV se sobreexpresa en las células sf9, basado en el calor basado en el choque pDHsp/V5-suSpodoptera frugiperda Handy sistema (células sf9). PDHsp/V5-su contenido Drosophila calor choque proteína promotora, que impulsa la expresión del gen aguas abajo a una temperatura de 42 ° C condición mediante el uso de factores transcripcionales celulares y la traducción del sistema (<strong...

Watch this Scientific Journal Video about Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay at JoVE.com

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

Este protocolo describe un calor proteína inducida por choque expresión sistema (pDHsp/V5-su/sf9 de la célula), que puede utilizarse para evaluar la actividad anti-apoptótica de posibles proteínas extrañas y su truncado amino o expresando proteínas extrañas ácidos en las células de los insectos.

Expresión transitoria de Genes foráneos en células de insecto (sf9) para el análisis funcional de proteínas

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

The overall goal of this heat shock-induced protein expression system is to evaluate protein anti-apoptotic activity in two potentially functionally antagonistic proteins in insect cells. This method can help answer key questions about the functions and activities of candidate proteins in biotechnology and proteomics, such as their anti-apoptotic activities and protein interactions. The main advantage of this technique is that the timing of gene expression is controlled by heat shock induction.So this method can provide insight into protein functional status. It can also be applied to other systems, such as mammalian protein expression. The Spodoptera frugiperda, or Sf9, insect cells used in this protocol are grown in cell culture medium in 25 square centimeter cell culture flasks at 28 degrees Celsius.Shake the cell culture flask to detach the cells from the flask. Then check under a light microscope to make sure that about 80%of the cells are detached. Transfer 50%of the cell suspension to a new 25 square centimeter cell culture flask, and allow the cells to attach at room temperature for 15 minutes.After 15 minutes, replace the medium with five milliliters of fresh culture medium, and grow in an incubator at 28 degrees Celsius. Depending on the cell growth, passage the cells every two to three days. Prior to harvesting the Sf9 cells, it is important to check the cells under the light microscope to make sure that there is no contamination by bacteria or other microorganisms.To harvest the Sf9 cells, shake the culture flask to detach the cells, and then transfer the cell suspension to a 15-milliliter tube. Using a P10 pipette, transfer 10 microliters of the cell suspension to a hemacytometer, and count the cell number under the light microscope. Plate three times 10 to the fifth cells into each well of a 24-well plate, and leave at room temperature for 15 minutes.After 15 minutes, replace the medium with 0.5 milliliters of plating medium. Prepare the cell transfection regent. For each transfection, dilute eight microliters of cell transfection reagent with 100 microliters of serum-free cell culture medium, and vortex for one second to mix.In this study, three different truncations of the baculoviral gene, inhibitor of apoptosis 3, or IAP3, will be expressed under the control of the Drosophila heat shock 70 promoter, the full-length IAP3, a truncation lacking the RING domain, and a truncation lacking the BIR domain. Add two micrograms of each plasmid DNA into 100 microliters of serum-free cell culture medium, and mix by vortexing for one second. Prepare a positive control as well.Combine the diluted plasmid DNA and diluted cell transfection reagent, and mix by vortexing. Incubate at room temperature for 30 minutes. Using a P1000 pipette, add 210 microliters of each DNA transfection reagent mixture dropwise onto the cells.Incubate at 28 degrees Celsius for five hours. After five hours, replace the plating medium with 0.5 milliliters of cell culture medium, and then seal the 24-well plate with tape. Incubate at 28 degrees.On the following day, heat shock to induce protein expression by floating the plate in a 42 degree Celsius water bath for 30 minutes. After 30 minutes, return the 24-well plate to the 28 degree Celsius incubator. Protein expression is subsequently confirmed by Western blot as described in the text protocol.To examine gene-induced cell apoptosis, first prepare Sf9 cells as demonstrated earlier. Next, transfect the cells in the same manner as before, but include one microgram of a plasmid containing an apoptosis inducer gene in each transfection reaction. Incubate at 28 degrees Celsius for five hours, replace the medium, and incubate for 16 hours.Heat shock the transfected cells at 42 degrees Celsius for 30 minutes. Incubate the cells at 28 degrees Celsius for five hours before conducting the cell viability assay. For chemical-induced cell apoptosis, plate one times 10 to the six Sf9 cells into each well of a six-well plate.Leave at room temperature for 15 minutes, and then replace the medium with one milliliter of plating medium. Transfect the cells with four micrograms of each plasmid DNA. After five hours, replace the plating medium with one milliliter of cell culture medium, and incubate the cells at 28 degrees Celsius for 16 hours.Then heat shock at 42 degrees Celsius for 30 minutes. Incubate the cells at 28 degrees Celsius for five hours post-heat shock. Then treat the transfected cells with actinomycin D to induce apoptosis.Incubate at 28 degrees Celsius for 16 hours before conducting the cell viability assay. Begin this procedure by washing the treated cells. Remove the medium from each well, add one milliliter of PBS, and leave for one minute.Wash the cells three times with PBS in this manner. Resuspend the cells in each well with one milliliter of PBS containing 04%trypan blue, and stain at room temperature for three minutes. Transfer the cell suspension into a 1.5 milliliter microfuge tube.Transfer 10 microliters of the trypan blue-stained cell suspension to a hemacytometer, and count the viable intact cells under a light microscope. Finally, perform statistical analysis as described in the text protocol. The full length and two truncations of IAP3 from Lymantria xylina MNPV were overexpressed in Sf9 cells, and Western blot was performed on cell lysates to confirm protein expression.The anti-apoptotic protein Ac-P35 was included as a positive control. All proteins could be detected in transfected cells at either one hour or five hours post-heat shock, with a higher expression at five hours. This system can be adapted to evaluate anti-apoptotic activity.For gene-induced cell apoptosis, the apoptosis inducer Drosophila Reaper was co-transfected with target gene plasmid constructs into Sf9 cells, which were subsequently heat shocked to activate gene expression. Compared to the vector Drosophila Reaper, the positive control showed high anti-apoptotic activity, reaching up to 80%of the viability rate. The other constructs showed various effects on anti-apoptotic activity.For chemical-induced cell apoptosis, only one construct was transfected into Sf9 cells, and actinomycin D was added five hours post-heat shock. Compared to the positive control or the negative control, the other constructs showed various effects on anti-apoptotic activity. Once mastered, this technique can be done in two to three days if it is performed properly.While attempting this procedure, it's important to remember to obey the operation rules of cell culture in laminar flow and check the status of cells diligently. Following this procedure, other methods, such as co-immunoprecipitation, can be performed to answer additional questions, like whether there is any interaction between the two proteins co-expressed in the insect cells. After its development, this technique paved the way for researchers in the field of biotechnology to explore protein functions in animal or microorganisms.After watching this video, you should have a good understanding of how to use this system to express foreign protein or evaluate potentially functionally antagonistic proteins in insect cells. Don't forget that working with some chemicals, like actinomycin D, can be extremely hazardous, and precautions, such as wearing gloves, should always be taken while performing this procedure.

Research

JoVE Journal

Genetics

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

Controllable Ion Channel Expression through Inducible Transient Transfection

Expresión Ion Channel controlable a través de la transfección transitoria inducible

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Investigación

Genética

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

In Vitro Ensayo de bioluminiscencia para la caracterización del ritmo circadiano en células epiteliales mamarias

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Determinación de la óptima Localización cromosómica de un elemento de DNA en Escherichia coli utilizando un nuevo enfoque mediada por transposones

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

Mantenimiento de cultivos biológicos y medir la expresión génica en Aphis nerii: un sistema no-modelo de las interacciones planta-insecto

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

Proteína endógena Tagging en humanos inducida por células madre pluripotentes utilizando CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

Reverse Transcription-Loop-mediated Isothermal Amplification (RT-LAMP) Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

Reverse Transcription-Loop-mediated Isothermal Amplification RT-LAMP Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

Revertir el ensayo de amplificación isotérmica mediada por el lazo de transcripción (RT-LAMP) para el Virus Zika y Housekeeping Genes en muestras de mosquitos, suero y orina

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Identificación de eventos de recombinación homóloga en células madre embrionarias del ratón mediante Southern blot y reacción en cadena de polimerasa

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

Determinar la tasa de fertilización de huevos de Bemisia tabaci mediante una técnica citogenética

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

Un ensayo para cuantificar la unión de proteínas y ARN en bacterias

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

Un protocolo in vitro para evaluar los niveles, funciones y genes objetivo asociados de MicroRNA en células tumorales

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...