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

Si ringrazia il Dr. Jian-Horng Leu dell'Istituto di Biologia Marina, National Taiwan Ocean University per la fornitura di 3 costruzioni di plasmide. Questa ricerca è stata sostenuta da Grant 106-2311-B-197-001 - dal Ministero della scienza e della tecnologia (MOST).

1. preparati
<ol><li>Coltura delle cellule dell'insetto<ol>
<li>Preparare 50 mL di mezzo di coltura cellulare. A tale scopo, aggiungere 500 µ l di antibiotici (anfotericina B = 0,25 µ g/mL, penicillina = 100 unità/mL, streptomicina = 100 µ g/mL) e 5 mL di siero bovino fetale inattivati al calore nel mezzo di coltura privo di siero cellulare (senza FBS o antibiotici). Nota: Riscaldare il siero bovino fetale a 65 ° C per 30 min in un bagno di acqua prima dell'uso.</li>
		<li>Mantenere Spodopt...

<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>

Il concetto di sistema basati su shock cellulare pDHsp/V5-His/sf9 calore era in primo luogo descritto da Clem et nel 19948. Confronto del promotore del gene di baculovirus (IE1) e Drosophila hsp70 ha mostrato che quel hsp70 aveva una maggiore efficienza nella zanzara cellule10. Inoltre, a causa dell'induzione di scossa di calore, la tempistica dell'espressione della proteina potrebbe essere controllata precisamente dopo trattamento di sho...

Due sistemi di espressione della proteina sono stati comunemente utilizzati per la produzione di proteine: sistemi di espressione proteica prokaryote (sistema di espressione del gene diEscherichia coli ) e sistemi di espressione della proteina dell'eucariota. Un sistema di espressione della proteina eucariote popolare è il baculovirus espressione vettoriale sistema (BEVS)1. Baculoviridae in primo luogo sono stati usati come agenti di controllo biologico in tutto il mondo dell'agricoltura e della foresta di parassiti. Negli ultimi decenni, Baculoviridae sono stati sviluppati come strumenti biotecnologici per vettori di espressione di proteine pure. I genomi di Baculoviridae sono costituiti da DNA circolare a doppia elica e avvolti i nucleocapsids2. Ad oggi, più di settantotto baculovirus isolati sono state sequenziate3. Basato sulla cascata temporale di baculovirus espressioni di gene in cellule di insetto ospitante, trascrizione genica poteva essere classificata in quattro cascate temporale, compreso immediati-presto, geni di in ritardo-inizio, fine e molto fine4.
BEVSs sono stati indicati in modo che i promotori di geni molto tardi (cioè, poliedro o p10 promotore) sono stati usati per guidare i geni bersaglio, mentre il baculovirus ricombinanti è stato generato da una ricombinazione omologa. Espressione delle proteine straniere in cellule di insetto di baculovirus ricombinanti è simile a quella delle proteine dei mammiferi a modificazioni post-traduzionali (adatti per la produzione di glicoproteina). Così, il baculovirus è stato ampiamente usato5,6,7. Tuttavia, una limitazione è la presenza di vie di N-glicosilazione differenti in cellule di insetto7.
Di conseguenza, un nuovo sistema di espressione di baculovirus, il sistema di espressione del gene transitoria, è stato sviluppato. Questo sistema esprime geni estranei sotto l'unità di baculovirus immediati-presto promotori (promotore diie-1 ) in cellule di insetto. Utilizzando questo sistema, la proteina bersaglio può essere espresso immediatamente sotto il controllo del promotore ie-1 durante la modifica la via di N-glicosilazione in cellule di insetto, con conseguente migliore oligosaccaridi N-collegati7. Inoltre, geni immediati-presto baculovirus sono trascritti dalla RNA polimerasi II cellula ospite e non richiedono alcun fattore virale per attivazione4. Di conseguenza, proteine straniere possono essere espresso in cellule di insetto in breve tempo. Ad oggi, il transitorio sistema di espressione genica è una delle tecnologie più importanti per l'esecuzione di saggi funzionali della proteina in baculovirus in vitro sistema di coltura cellulare. Il sistema può essere applicato per analizzare la funzione di baculovirus o proteine straniere. Uno dei sistemi di espressione genica transitoria commercialmente disponibile si basa sui promotori di geni immediati-presto (IE) di Orgyia pseudotsugata virus nucleopolyhedrovirus (OpMNPV) (OpIE2 e OpIE1 promotori).
Tuttavia, il livello inferiore di espressione di geni estranei in cellule di insetto era ancora un problema quando il sistema di espressione del gene transitoria basata sul promotore di OpIE è stato usato8,9,10. Così, un altro sistema di espressione genica transitoria è stato costruito in base il promotore della drosofila calore shock protein 70 (hsp70) gene di8,9. Il promotore di hsp70 funziona più efficientemente di baculovirus IE promotore quando indotta da shock termico in cellule di insetto10. In questo sistema, sono stati espressi i geni bersaglio sotto l'unità del promotore di Drosophila heat shock 70 (Dhsp70). Geni estranei possono essere facilmente clonati nel plasmide di espressione genica transitoria con i metodi di clonazione PCR-basati. Inoltre, il controllo di temporizzazione per l'espressione genica può essere eseguito tramite induzione di scossa di calore.
In questo rapporto, seguiamo l'approccio ed esprimere tre diversi troncamenti del gene di baculovirus (inibitore dell'apoptosi 3, iap3 da Lymantria xylina MNPV) utilizzando il sistema di espressione basati su scossa transitoria della proteina di calore e applicare ulteriormente queste proteine espresse sulla analisi delle attività anti-apoptotica. Questo sistema può sia esprimere proteine straniere rapidamente o essere applicato alla valutazione delle attività anti-apoptotica della proteina in cellule sf9, pur avendo anche il potenziale per essere applicato ad altre analisi di attività della proteina.

<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

Il sistema di espressione genica transitoria è una delle tecnologie più importanti per l'esecuzione di analisi funzionale della proteina in baculovirus in vitro sistema di coltura cellulare. Questo sistema è stato sviluppato per esprimere stranieri geni sotto il controllo del promotore baculovirus in plasmidi di espressione transiente. Inoltre, questo sistema può essere applicato a un'analisi funzionale del baculovirus sé o proteine straniere. Il sistema di espressione genica transitoria più ampiamente e commercialmente disponibile è sviluppato sul promotore del gene immediati-presto (IE) di Orgyia pseudotsugata virus nucleopolyhedrovirus (OpMNPV). Tuttavia, è stato osservato un livello basso di espressione di geni estranei in cellule di insetto. Di conseguenza, un sistema di espressione genica transitoria è stato costruito per migliorare l'espressione della proteina. In questo sistema, plasmidi ricombinanti sono stati costruiti per contenere la sequenza di destinazione sotto il controllo del promotore (Dhsp70) 70 di Drosophila calore shock. Questo protocollo presenta l'applicazione di questo calore shock-base pDHsp/V5-His (epitopo V5 con 6 istidina) /Spodoptera frugiperda cell sistema (sf9 cella); Questo sistema è disponibile non solo per l'espressione genica ma anche per valutare l'attività anti-apoptotica di candidato proteine in cellule di insetto. Inoltre, questo sistema può essere sia transfected con un plasmide ricombinante o co-trasfettate due plasmidi ricombinanti potenzialmente funzionalmente antagonistiche in cellule di insetto. Il protocollo dimostra l'efficienza di questo sistema e fornisce un caso pratico di questa tecnica.

L'intera lunghezza e altri due troncamenti (domini BIR e anello) di Ly-IAP3 da LyxyMNPV overexpressed in cellule sf9, basato sul calore shock-base pDHsp/V5-His /Spodoptera frugiperda (sf9 cella) sistema di celle. Il pDHsp/V5-His conteneva un promotore di un proteina di scossa calore di Drosophila , che aziona l'espressione genica a valle ad una temperatura di 42 ° C condizione utilizzando fattori trascrizionali cellulari e la traduzione del sistema (<strong class="xfig"...

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

Questo protocollo descrive un sistema di espressione di proteina indotta da scossa di calore (sistema pDHsp/V5-His/sf9 cellulare), che può essere utilizzato per esprimere proteine straniere o per valutare l'attività anti-apoptotica di potenziali proteine straniere e loro troncato amminico acidi in cellule di insetto.

Espressione transiente di geni estranei in cellule di insetto (sf9) per analisi funzionale della proteina

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

Controllabile Espressione Ion Canale attraverso inducibile trasfezione transiente

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

Ricerca

Genetica

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 Dosaggio di bioluminescenza per caratterizzare il ritmo circadiano in cellule epiteliali mammarie

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

Determinazione dei percorsi cromosomici ottimali per un elemento di DNA in Escherichia coli usando un approccio novello Transposon-mediata

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

Mantenimento di colture biologiche e misurazione dell'espressione genica in Aphis nerii: un sistema Non-modello per le interazioni pianta-insetto

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

Proteina endogena Tagging in umani hanno indotto le cellule staminali pluripotenti utilizzando 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

Invertire l'amplificazione isotermica mediata da Loop-trascrizione (RT-LAMP) analisi per Virus di Zika e geni Housekeeping in urina, siero ed esempi di zanzara

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

Identificazione di eventi di ricombinazione omologa in cellule staminali embrionali del Mouse mediante Southern Blotting e reazione a catena della polimerasi

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

Determinare il tasso di fertilizzazione dell'uovo di Bemisia tabaci utilizzando una tecnica citogenetica

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 saggio per quantificare il legame proteino-RNA nei batteri

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 protocollo in vitro per la valutazione dei livelli, delle funzioni e dei geni target associati nelle cellule tumorali

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