We thank Dr. Jian-Horng Leu of Institute of Marine Biology, National Taiwan Ocean University for providing 3 plasmid constructions. This research was supported by Grant 106-2311-B-197 -001 - from the Ministry of Science and Technology (MOST).

1. Preparations
<ol>
	<li>Insect cell culture
	<ol>
		<li>Prepare 50 mL of cell culture medium. To do so, add 500 &#181;L of antibiotics (Amphotericin B = 0.25 &#181;g/mL, Penicillin = 100 unit/mL, Streptomycin = 100 &#181;g/mL) and 5 mL of heat-inactivated fetal bovine serum in serum-free cell culture medium (without FBS or antibiotics). 
		NOTE: Heat the fetal bovine serum at 65 &#176;C for 30 min in a water bath before use.</li>
		<li>Maintain Spodoptera frugiperda (Lepidoptera: Noctuidae), sf9 insect cells. Detach ca. 80% of the cells from 25 cm2 cell culture flask by shaking the flask and check under light microscopy. Then, transfer 50% of the cell suspension to a new 25 cm2 cell culture flask and allow the cells to attach for 15 min at room temperature. Replace the medium with 5 mL fresh cell culture medium and grow in an incubator at 28 &#176;C. Passage cells every 2 to 3 days depending on the cell growth.</li>
	</ol>
	</li>
	<li>Preparation of plasmids for cell transfection
	<ol>
		<li>Insert the target DNA fragments (e.g., Lymantria xylina MNPV (LyxyMNPV) iap3 gene and its deletion constructs) into pDHsp/V5-His by PCR-based cloning method11. Check the growth of transformed colonies by colony PCR using PCR Master Mix (2X) and the pDhsp-F2/Op-IE2R primer set. Confirm the plasmid sequences by commercial sequencing service. 
		NOTE: Table 1 lists the primers used for PCR and the corresponding constructs.</li>
		<li>Culture single sequenced bacterial colonies, which contain the aforementioned plasmid constructions (step 1.2) in 200 mL LB medium containing selected antibiotics (50 &#181;g/mL), respectively.</li>
		<li>Extract the plasmids from the cultured E. coli using Midi Plasmid Kit according to manufacturer&#39;s instructions12.</li>
	</ol>
	</li>
	<li>Prepare plating medium by mixing 1.5 mL of cell culture medium and 8.5 mL of serum-free cell culture medium.</li>
	<li>Prepare Actinomycin D (ActD) cell culture medium by adding 1.5 &#181;L of ActD stock (1 mg/mL) into 10 mL of cell culture medium (final concentration = 150 ng/mL). Store at 4 &#176;C.</li>
</ol>
2. Protein transient expression
<ol>
	<li>Cell seeding
	<ol>
		<li>Harvest the sf9 cells by shaking culture flask, topple and fall the cell suspension to 50 mL tube, and transfer 10 &#181;L to hemocytometer by a P10 pipette. Count the cell number under a light microscope.</li>
		<li>Plate 3 &#215; 105 sf9 cells into each well in a 24-well plate for 15 min at room temperature. Replace the medium with 0.5 mL of plating medium.</li>
	</ol>
	</li>
	<li>Transfection of plasmids
	<ol>
		<li>Dilute cell transfection reagent: Dilute 8 &#181;L of cell transfection reagent in 100 &#181;L serum-free cell culture medium and mix by vortexing for 1&#160;s.</li>
		<li>Add 2 &#181;g of plasmid DNA (pDHsp70-Ac-P35/V5-His or pDHsp70-Ly-IAP3/V5-His or pDHsp70-Ly-IAP3-BIR/V5-His or pDHsp70-Ly-IAP3-RING/V5-His) into 100 &#181;L of serum-free cell culture medium and mix by vortexing for 1 s (Figure 2).</li>
		<li>Combine the diluted plasmid DNA and diluted cell transfection reagent (210 &#181;L), and mix by vortexing for 1 s. Incubate for 30 min at room temperature.</li>
		<li>Add 210 &#181;L of DNA transfection reagent mixture dropwise onto the cells by a P1000 pipette. Incubate at 28 &#176;C for 5 h.</li>
		<li>Replace the plating medium with 0.5 mL of cell culture medium using a P1000 pipette. Seal the 24-well plate with tape and incubate the cells at 28 &#176;C for 16 h.</li>
	</ol>
	</li>
	<li>Heat shock the transfected cells: Put the plate in a 42 &#176;C water bath (floating on the water surface). Heat for 30 min and return the 24-well plate to the 28 &#176;C incubator.</li>
	<li>Detection of protein expression
	<ol>
		<li>After 1 h or 5 h heat shock,wash the cells with 0.5 mL of 1x&#160;PBS buffer briefly three times. 
		NOTE: Dilute 10x&#160;PBS buffer to 1x PBS buffer by adding 1 mL of 10x&#160;PBS buffer to 9 mL sterilized ddH2O</li>
		<li>Lyse the cells with 40 &#181;L of 1x&#160;SDS Loading Dye by pipetting up and down. 
		NOTE: Dilute 4x&#160;SDS Loading Dye by mixing 30 &#181;L of 1x&#160;PBS buffer and 10 &#181;L of 4x&#160;SDS Sample Buffer.</li>
		<li>Heat the protein samples at 98 &#176;C for 10 min in a heat block, spin down for 1 min, and put on ice for Western blot assay.</li>
		<li>Western blot assay 
		Follow the procedure of Western blot assay from Eslami and Lujan13. Run SDS-PAGE gels14: one gel subjected to Coomassie blue staining (loading control for checking that the quantity of protein samples loaded in each well is equal in amount) and the other subjected to Western blot assay, according to Eslami and Lujan13.</li>
		<li>Detect V5-tagged fusion proteins with rabbit anti-V5 antibody (5 mg/mL) (1:5000 dilution in TBST buffer to working concentration 1 &#181;g/mL) and goat anti-rabbit IgG-horseradish peroxidase (HRP) conjugate (0.8 mg/mL) (1:10000 dilution in TBST buffer to working concentration 0.08 &#181;g/mL). 
		NOTE: Adjust the percentage of polyacrylamide to 17.5% when the protein molecular weight to be &#60;17 kDa.</li>
	</ol>
	</li>
</ol>
3. Anti-apoptotic activity assay
<ol>
	<li>Gene-induced cell apoptosis: Repeat the aforementioned procedure from 2.1 to 2.3. Co-transfect 1 &#181;g of pDHsp/D-rpr/FLAG-His plasmid DNA (containing apoptosis inducer gene) with 1 &#181;g of plasmid DNA [pDHsp70/V5-His vector (negative control), pDHsp70-Ac-P35/V5-His (positive control), pDHsp70-Ly-IAP3/V5-His, pDHsp70-Ly-IAP3-BIR/V5-His or pDHsp70-Ly-IAP3-RING/V5-His, respectively.]. At 5 h post-heat shock treatment, conduct the cell viability assay (Figure 2).</li>
	<li>Chemical-induced cell apoptosis: Repeat the aforementioned procedure from 2.1 to 2.3. In step 2.1.2, plate 1 x 106 sf9 cells into each well in a 6-well plate. Transfect 4 &#181;g of plasmid DNA [pDHsp70/V5-His vector (negative control), pDHsp70-Ac-P35/V5-His (positive control), pDHsp70-Ly-IAP3/V5-His, pDHsp70-Ly-IAP3-BIR/V5-His or pDHsp70-Ly-IAP3-RING/V5-His, respectively.]. At 5 h post-heat shock, treat sf9 cells with 2mL of ActD cell culture medium for 16 h and conduct the cell viability assay (Figure 2). 
	NOTE: Minimum volume to cover one&#160; well of 6-well plate is 1 mL.</li>
	<li>Perform the above anti-apoptotic activity assay experiments, including 3.1 and 3.2 in triplicates.</li>
</ol>
4. Cell viability assay
<ol>
	<li>Wash the treated cells by adding 1 mL of 1x&#160;PBS buffer for 1 min 3 times. Resuspend the cells by pipetting 1 mL 1x&#160;PBS buffer containing 0.04% trypan blue and stain for 3 min at room temperature. Transfer the cell suspension into a 1.5 mL microtube. 
	NOTE: Dilute 0.4% trypan blue solution by mixing 9 mL of 1x&#160;PBS buffer and 1 mL 0.4% trypan blue solution.</li>
	<li>Transfer 10 &#181;L trypan blue-stained cell suspension to the hemocytometer with a P10 pipette and count the viable intact cells under a light microscope.</li>
	<li>Statistical analysis
	<ol>
		<li>Calculate the recorded data and present as the means &#177; S.D. for all counts.</li>
		<li>Analyze the data using the Student&#39;s two-tailed t-test by with Microsoft Excel. Define statistically significant data as P-value &#60;0.05.</li>
	</ol>
	</li>
</ol>

<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://jove.unicartagenaproxy.elogim.com/science-education/5058/separating-protein-with-sds-page">https://jove.unicartagenaproxy.elogim.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>

The authors declare that they have no competing financial interests.

The concept of heat shock-based pDHsp/V5-His/sf9 cell system was first described by Clem et al. in 19948. Comparison of the baculoviral gene promoter (IE1) and Drosophila hsp70 showed that hsp70 had a higher efficiency in mosquito cells10. Furthermore, due to the heat shock induction, the timing of protein expression could be controlled precisely after heat shock treatment. This system was then applied for protein functional assays of shr...

Two protein expression systems have been commonly used for producing proteins: prokaryote protein expression systems (Escherichia coli gene expression system) and eukaryote protein expression systems. One popular eukaryote protein expression system is the baculovirus expression vector system (BEVS)1. Baculoviruses were first used as worldwide biological control agents of agricultural and forest pests. In the last few decades, baculoviruses were developed as biotechnological tools for protein expression vectors as well. The genomes of baculoviruses consist of double-stranded circular DNA and enveloped nucleocapsids2. To date, more than seventy-eight baculovirus isolates have been sequenced3. Based on the temporal cascade of baculoviral gene expressions in host insect cells, the gene transcription could be classified into four temporal cascades, including immediate-early, delayed-early, late, and very late genes4.
BEVSs were designated so that the very late gene promoters (i.e., polyhedron or p10 promoter) were used to drive the target genes, while the recombinant baculovirus was generated by homologous recombination. Expression of foreign proteins in insect cells by recombinant baculovirus is similar to that of mammalian proteins in post-translational modifications (suited for glycoprotein production). Thus, the baculovirus has been widely used5,6,7. However, one limitation is the presence of different N-glycosylation pathways in insect cells7.
Therefore, a new baculovirus expression system, the transient gene expression system, was developed. This system expresses foreign genes under the drive of baculoviral immediate-early promoters (ie-1 promoter) in insect cells. By using this system, the target protein can be immediately expressed under the control of ie-1 promoter while modifying the N-glycosylation pathway in insect cells, resulting in better N-linked oligosaccharides7. Moreover, baculoviral immediate-early genes are transcribed by the host cell RNA polymerase II and do not require any viral factor for activation4. Therefore, foreign proteins can be expressed in insect cells within a short time. To date, the transient gene expression system is one of the most important technologies for performing protein functional assays in the baculovirus in vitro cell culture system. The system can be applied to analyze the function of either baculovirus or foreign proteins. One of the commercially available transient gene expression systems is based on the immediate-early gene (IE) promoters of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) (OpIE2 and OpIE1 promoters).
However, the lower expression level of foreign genes in insect cells was still a problem when the OpIE promoter-based transient gene expression system was used8,9,10. Thus, another transient gene expression system was constructed based on the promoter of the Drosophila heat shock protein 70 (hsp70) gene8,9. The promoter of hsp70 works more efficiently than baculoviral IE promoter when induced by heat shock in insect cells10. In this system, the target genes were expressed under the drive of Drosophila heat shock 70 (Dhsp70) promoter. Foreign genes can be easily cloned into the transient gene expression plasmid by PCR-based cloning methods. Furthermore, the control of timing for gene expression can be performed by heat shock induction.
In this report, we follow the approach and express three different truncations of the baculoviral gene (inhibitor of apoptosis 3, iap3 from Lymantria xylina MNPV) by using the heat shock-based transient protein expression system and further apply these expressed proteins on anti-apoptotic activity analysis. This system can either express foreign proteins quickly or be further applied to the evaluation of protein anti-apoptotic activity in sf9 cells, while also having the potential to be applied to other protein activity assays.

<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

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell culture system. This system was developed to express foreign genes under the control of the baculoviral promoter in transient expression plasmids. Furthermore, this system can be applied to a functional assay of either the baculovirus itself or foreign proteins. The most widely and commercially available transient gene expression system is developed based on the immediate-early gene (IE) promoter of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). However, a low expression level of foreign genes in insect cells was observed. Therefore, a transient gene expression system was constructed for improving protein expression. In this system, recombinant plasmids were constructed to contain the target sequence under the control of the Drosophila heat shock 70 (Dhsp70) promoter. This protocol presents the application of this heat shock-based pDHsp/V5-His (V5 epitope with 6 histidine)/Spodoptera frugiperda cell (sf9 cell) system; this system is available not only for gene expression but also for evaluating the anti-apoptotic activity of candidate proteins in insect cells. Furthermore, this system can be either transfected with one recombinant plasmid or co-transfected two potentially functionally antagonistic recombinant plasmids in insect cells. The protocol demonstrates the efficiency of this system and provides a practical case of this technique.

The full length and other two truncations (BIR and RING domains) of Ly-IAP3 from LyxyMNPV were overexpressed in sf9 cells, based on the heat shock-based pDHsp/V5-His/Spodoptera frugiperda cell (sf9 cell) system. The pDHsp/V5-His contained a Drosophila heat shock protein promoter, which drives downstream gene expression at a temperature of 42 &#176;C condition by using cellular transcriptional factors and the translation system (Figure 1)<sup...

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

This protocol describes a heat shock-induced protein expression system (pDHsp/V5-His/sf9 cell system), which can be used for either expressing foreign proteins or evaluating the anti-apoptotic activity of potential foreign proteins and their truncated amino acids in insect cells.

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

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

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12.2K Views.  National Ilan University. This protocol describes a heat shock-induced protein expression system (pDHsp/V5-His/sf9 cell system), which can be used for either expressing foreign proteins or evaluating the anti-apoptotic activity of potential foreign proteins and their truncated amino acids in insect cells.

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

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

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

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

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

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

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

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

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms to questions surrounding insect's interactions with their host plants. Genomic resources are available for several aphid species, and with advances in the next-generation sequencing, transcriptomic studies are being extended to non-model organisms that lack genomes. Furthermore, aphid cultures can be collected from the field and reared in the laboratory for the use in organismal and molecular experiments to bridge the gap between ecological and genetic studies. Last, many aphids can be maintained in the laboratory on their preferred host plants in perpetual, parthenogenic life cycles allowing for comparisons of asexually reproducing genotypes. Aphis nerii, the milkweed-oleander aphid, provides one such model to study insect interactions with toxic plants using both organismal and molecular experiments. Methods for the generation and maintenance of the plant and aphid cultures in the greenhouse and laboratory, DNA and RNA extractions, microsatellite analysis, de novo transcriptome assembly and annotation, transcriptome differential expression analysis, and qPCR verification of differentially expressed genes are outlined and discussed here.

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

The aphid Aphis nerii colonizes on highly-defended plants in the dogbane family (Apocyanaceae) and provides numerous opportunities to study plant-insect interactions. Here, we present a series of protocols for the maintenance of plant and aphid cultures, and the generation and analysis of molecular and -omic data for A. nerii.

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

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.

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

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth defects. The goal of this protocol is to quickly detect ZIKV in both human and mosquito samples. The current gold standard for ZIKV detection is quantitative reverse transcription PCR (qRT-PCR); reverse transcription loop-mediated isothermal amplification (RT-LAMP) may allow for a more efficient and low-cost testing without the need for expensive equipment. In this study, RT-LAMP is used for ZIKV detection in various biological samples within 30 min, without first isolating the RNA from the sample. This technique is demonstrated using ZIKV infected patient urine and serum, and infected mosquito samples. 18S ribosomal ribonucleic acid and actin are used as controls in human and mosquito samples, respectively.

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

This protocol provides an efficient and low-cost method to detect Zika virus or control targets in human urine and serum samples or in mosquitoes by reverse transcription loop-mediated isothermal amplification (RT-LAMP). This method does not require RNA isolation and can be done within 30 min.

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

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 editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

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

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 viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

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

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

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 ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame (ORF) annotation impose two arbitrary criteria: a minimum length of 100 codons and a single ORF per transcript. However, a growing number of studies report expression of proteins from allegedly non-coding regions, challenging the accuracy of current genome annotations. These novel proteins were found encoded either within non-coding RNAs, 5' or 3' untranslated regions (UTRs) of mRNAs, or overlapping a known coding sequence (CDS) in an alternative ORF. OpenProt is the first database that enforces a polycistronic model for eukaryotic genomes, allowing annotation of multiple ORFs per transcript. OpenProt is freely accessible and offers custom downloads of protein sequences across 10 species. Using OpenProt database for proteomic experiments enables novel proteins discovery and highlights the polycistronic nature of eukaryotic genes. The size of OpenProt database (all predicted proteins) is substantial and need be taken in account for the analysis. However, with appropriate false discovery rate (FDR) settings or the use of a restricted OpenProt database, users will gain a more realistic view of the proteomic landscape. Overall, OpenProt is a freely available tool that will foster proteomic discoveries.

OpenProt is a freely accessible database that enforces a polycistronic model of eukaryotic genomes. Here, we present a protocol for the use of OpenProt databases when interrogating mass spectrometry datasets. Using OpenProt database for analysis of proteomic experiments allows for discovery of novel and previously undetectable proteins.

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

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

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3&#8217; untranslated regions (3&#8217; UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

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

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

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Chapters in this video

Controllable Ion Channel Expression through Inducible Transient Transfection

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

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

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

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

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

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

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

An Assay for Quantifying Protein-RNA Binding in Bacteria

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