Name: mission lentiplex pooled shrna library screening in mammalian cells
Uploaded: 2011-12-21T12:00:00
Duration: 9 min 12 s
Description: Watch this Scientific Journal Video about MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells at JoVE.com

MISSION is a registered trademark of Sigma-Aldrich Biotechnology L.P. 
LentiPlex is a trademark of Sigma-Aldrich Biotechnology L.P.

LentiPlex Pooled Screen Setup
To identify shRNA sequences of interest, it is critical to set up the screen such that the majority of cells receive only one shRNA construct. By using a low MOI (multiplicity of infection), the probability of multiple integrants per cell is greatly decreased. However, transduction efficiencies, and therefore desired MOIs, depend strongly on the target cell type. Therefore, it is imperative that determination of the optimal MOI is carried out before starting the screen in a new cell type.
1. Transduction Of Target Cells with the Mission LentiPlex Pooled shRNA Library 
<ol>
 <li>The actual MOI used will vary for different cell types. If replicates are desired then the number of plates must be increased accordingly. In addition, typically it will not be advantageous to combine the different subpools. Keeping the subpools separate will aid in the deconvolution process. Ensure proper labeling of each tissue culture dish with the subpool number that is applied during transduction. A low MOI is advised to reduce the probability of multiple integrants per cell. </li>
 <li>Day 1 &#x2013; Seed the appropriate number of 60-mm dishes with enough cells to obtain ~70% confluency on the following day (10 pools in total, each performed in triplicate = 30 dishes total). For our study, 60 mm dishes were seeded with 6 x 105 A549 cells to achieve a confluency of 70% prior to transduction. This ensures that cells are still in the exponential growth phase. Growth rates will vary by cell type and must be determined before starting the screen.</li>
 <li>Allow cells to adhere by incubating overnight at 37&deg;C in a humidified incubator in an atmosphere of 5% CO2.</li>
 <li>(Optional) Include two additional 60-mm dishes for transduction with negative control shRNAs. Label the dishes "Control A" for SHC001H and "Control B" for SHC002H.</li>
 <li>Day 2 &#x2013; Transduce the cells with the LentiPlex viral particles. On the day of transduction, thaw the lentiviral particles on ice.</li>
 <li>Transfer the thawed particles to a laminar flow hood and keep on ice if not being used immediately.</li>
 <li>Calculate how much of each individual viral sub-pool to add to the cells to get the MOI of 1 across all cell culture dishes.
 <ol>
 <li>Example: 1 x 106 cells/dish; Viral titer = 5 x 108 TU/ml; a desired MOI of 1. i.) 1 x 106 cells/dish x (MOI of 1) = 1 x 106 transducing units (TU) needed ii.) 1 x 106 TU/(5.0 x 108 TU/ml) obtained from the C of A = 2 &mu;L of lentiviral stock solution should be added to the appropriate dish. Dilute the calculated amount of the virus in 100-300 &mu;L of complete media in order to ensure better distribution of the virus when added to the cell culture dish.</li>
 </ol>
 </li>
 <li>Remove media from the pre-seeded cells. Add complete media containing hexadimethrine bromide solution to each dish. The recommended final concentration of hexadimethrine bromide in media is 8 &mu;g/ml. Use less hexadimethrine bromide if you find it to be toxic to your target cells.</li>
 <li>Add shRNA lentiviral particles to appropriate dishes in accordance with the predetermined experimental design. Gently swirl the plates to evenly distribute the virus across cells.</li>
 <li>Incubate 18-20 hours at 37&deg;C in a humidified incubator in an atmosphere of 5% CO2.</li>
 <li>(Optional) Repeat 3-8 at the identical MOI with SHC001H (empty vector shRNA Control) in Control Dish A and SHC002H (scrambled shRNA control) in Control Dish B. Treat these dishes identically to the experimental dishes throughout the experiment.</li>
 <li>Day 3 &#x2013; Change the media. Aspirate virus-containing media and add fresh complete media (without hexadimethrine bromide). Incubate the cells at 37&deg;C overnight.</li>
</ol>
2. Treat plated cells with therapeutic component/reagent
<ol>
 <li>Day 4 &#x2013; Aspirate media from wells and add fresh media containing therapeutic component at the optimal concentration. As with culture conditions, treatments will vary and proper concentrations and durations should be determined beforehand. In our study, we used 100 ng/mL of TRAIL and 1 &mu;M of Paclitaxel. Cells were treated for 48 hours. </li>
 <li>Surviving cells should be re-seeded into T75 flasks and allowed to expand until nearly 100% confluent.</li>
 <li>(Optional) At the end of selection, inspect Control Dish A and Control Dish B. Dish A and Dish B should each have fewer colonies than at least one of the pooled library dishes. If there are an unexpectedly high number of colonies in Dish A, than either the transduction process has affected a pathway related to the desired phenotype or the selective pressure isn't sufficiently strong and re-optimization of the assay may be required. If Dish B contains too many colonies, then expression of shRNA and engagement of the RNAi pathway may have an effect on the phenotype of interest. If this is the case repeat with SHC001H, to ensure that the effect isn't specific to SHC002H.</li>
</ol>
3. Deconvolution from a polyclonal cell population 
<ol>
 <li>Harvest the heterogeneous population of cells from each subpool and isolate genomic DNA. In our trial, cells were washed briefly in PBS and trypsinized before genomic DNA was isolated using the GenElute Mammalian Genomic Miniprep kit and supplied protocol (Sigma-Aldrich, G1N70). Alternatively, any other genomic purification kits that are currently available on the market can be used for DNA isolation. It is important to follow protocol recommendations for the starting amount of cultured cells.</li>
 <li>PCR amplification of targets. The shRNA template recovery procedure enables amplification of the entire pool of shRNA inserts from the selected cell population, or the retrieval of individual shRNA templates from colonies that were individually picked and expanded. After amplification of shRNA inserts from control and selected target cells, the PCR products can be sequenced.</li>
 <li>This PCR amplification step was optimized using Sigma ReadyMix, Catalog No. P2893. Other reagents can be used for amplification, but the cycling conditions and/or magnesium concentration may need to be modified for successful amplification. Amplification primers at the concentration 20 mM are included with the LentiPlex kit.</li>
 <li>Set up the PCR reaction as described in Table 1. Add the components in the order listed. The described amounts are for one 50 &mu;L reaction. For more reactions, scale the master mix components by the desired number of reactions and include 10% overage. The amount of DNA template recommended is 50-100 ng. </li>
 <li>It is strongly recommended that for one reaction the template consists of the MISSION shRNA Human Positive Control Vector diluted to the appropriate concentration. Successful amplification with this template indicates that the PCR assay components and cycling conditions are adequate. NOTE: To prevent carryover contamination of experimental samples and reagents, it is suggested that the positive control be diluted in a separate location from where subsequent PCR reactions are set up.</li>
 <li>Save the PCR program listed in Table 2 into your thermocycler.</li>
 <li>For each sample, combine 3 &mu;L PCR reaction with 1 &mu;L of dye and electrophorese alongside 5 &mu;L of DirectLoad&#x2122; PCR 100 bp Low Ladder, Catalog Number D3687 on a 1% agarose gel to confirm the presence of a 309 bp amplicon.</li>
 <li>Subcloning of PCR amplicons: PCR products were TOPO cloned using the TOPO-TA cloning kit, following the manufacturer's protocol (Invitrogen, K4575-01). The result is that one PCR product is contained in each vector.</li>
 <li>250 Individual bacterial colonies (each containing an individual PCR amplicon) were isolated and grown in 2 mL cultures of LB with 100 mg/mL ampicillin. Plasmid DNA was then extracted using the GenElute Plasmid Miniprep kit (Sigma-Aldrich, PLN70). </li>
 <li>Purified plasmid DNA was digested with EcoRI and electrophoresed to confirm the presence of the insert.</li>
 <li>Plasmid DNA samples were then sequenced and the shRNA insert identified using the LentiPlex shRNA Sequence Search database provided with the LentiPlex library.</li>
</ol>
4. Target Identification and Validation
<ol>
 <li>The shRNA sequence initiates with 5'-GAAACACCGG-3'. Enter at least the next 10 but less than 21 nucleotides as the query sequence into the Search box on the enclosed shRNA Sequence Search Access database form. </li>
 <li>Select the species of the pooled shRNA. Optionally, select the subpool from which the sequence was found. </li>
 <li>Now click on "Find Potential Hits" button to perform the search. This should identify the corresponding TRC shRNA sequence(s) that match the sequencing data. More information on the TRC shRNA sequence(s) identified and the corresponding gene targets can be readily found at Sigma-Aldrich's Your Favorite Gene: <a href="http://www.sigma.com/yfg">http://www.sigma.com/yfg</a></li>
 <li>Identified target genes should be validated by repeating the original screening assay with the original TRC clone plus at least two additional shRNAs that target the same gene using a 1 shRNA to 1 well format. True hits will display the same phenotype in a majority of wells.</li>
</ol>
5. Representative Results
An example of a LentiPlex pooled screen workflow is detailed in Figure 1. Transduced cells that proliferated in the presence of TRAIL and Paclitaxel were allowed to expand until flasks were confluent. Genomic DNA was harvested and subjected to the PCR amplification and cloning as illustrated in Figure 1 A, before being submitted for sequencing and shRNA identification as described in Figure 1 C. 250 clones were sequenced, of these 25 were represented by multiple clones and the remaining 225 were represented by only 1 clone and were not pursued at this time. 
As shown in Figure 2, several candidate genes including TBX3, PPP2CA, and AKT2 were represented by multiple clones. The T-box transcription factor, TBX3 appeared in a total of four clones and has been implicated in tumor migration5. PPP2CA downregulation has been demonstrated to maintain the growth of LNCaP cells cultured in androgen deficient conditions by relieving the androgen-deprivation-induced cell-cycle arrest and preventing apoptosis 6. AKT2 is a RAC-beta serine/threonine-protein kinase and putative oncogene demonstrated to play several roles in cancer development 7, 8.
 
<img src="/files/ftp_upload/3305/3305table1.jpg" alt="Table 1" /> 
*Final concentration of Mg2+ in solution will be 3 mM; 1.5 mM is contributed from the Taq ReadyMix and 1.5 mM from the Magnesium chloride solution. 
Table 1. Lentiplex PCR Reaction Conditions 
<img src="/files/ftp_upload/3305/3305table2.jpg" alt="Table 2" /> 
Table 2. Lentiplex PCR Amplification Program 
<img src="/files/ftp_upload/3305/3305fig1a.jpg" alt="Figure 1a" /> 
<img src="/files/ftp_upload/3305/3305fig1b.jpg" alt="Figure 1b" /> 
<img src="/files/ftp_upload/3305/3305fig1c.jpg" alt="Figure 1c" /> 
Figure 1. (A) LentiPlex pooled screen, (B) Polyclonal deconvolution workflow, and (C) identification of shRNA targets.
A. LentiPlex pooled screen. First, Seed cells, then add shRNA subpools, (10 pools in total, each performed in triplicate, for 30 dishes total). Next, treat with therapeutic component/reagent (in our case, TRAIL and Paclitaxel, respectively). Then, reseed surviving cells in flasks and allow to expand. Harvest heterogeneous population of cells from each subpool and isolate genomic DNA.
B. Polyclonal deconvolution. Perform PCR to amplify DNA containing the shRNA insert. The PCR product is uniform in size, but polyclonal in sequence since the template gDNA was also polyclonal. PCR product is cloned into the vector and then transformed into competent bacteria.
C. Identification of shRNA targets. Individual colonies are isolated and plasmid DNA is extracted. Each clone contains the cloning vector with one individual PCR fragment. Plasmid DNA is digested to confirm the presence of the insert and sequenced. The shRNA insert is identified using the LentiPlex shRNA Sequence Search database.
<img src="/files/ftp_upload/3305/3305fig2.jpg" alt="Figure 2" /> 
Figure 2. Identified Candidate Genes. 25 candidate genes were identified that had multiple hits. 225 candidate genes had only 1 hit and were not pursued. Please see Supplementary Table 1 for a breakdown of the individual clones per candidate gene.
<img src="/files/ftp_upload/3305/3305suptable1.jpg" alt="Table 1" /> 
Supplementary Table 1. Individual TRC Library clones Identified From Polyclonal Sequencing Gene ID Hits Detected Clones.

<ol>
	<li>Rao, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=siRNA+vs.+shRNA:+similarities+and+differences.">siRNA vs. shRNA: similarities and differences.</a> Adv. Drug Deliv. Rev. 61 (9), 746-746 (2009).</li><li>Root, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-scale+loss-of-function+screening+with+a+lentiviral+RNAi+library.">Genome-scale loss-of-function screening with a lentiviral RNAi library.</a> Nat Methods. 3 (9), 715-715 (2006).</li><li>Fan, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+antitumor+activity+of+TRAIL+combined+with+chemotherapeutic+agents+in+A549+cell+lines+in+vitro+and+in+vivo.">Synergistic antitumor activity of TRAIL combined with chemotherapeutic agents in A549 cell lines in vitro and in vivo.</a> Cancer Chemother Pharmacol. 55, 189-189 (2005).</li><li>Ji, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+screen+of+shRNAs+targeting+tumor+suppressor+genes+to+identify+factors+involved+in+A549+paclitaxel+sensitivity.">A screen of shRNAs targeting tumor suppressor genes to identify factors involved in A549 paclitaxel sensitivity.</a> Oncology Reports. 18, 1499-1499 (2007).</li><li>Fillmore, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+expands+breast+cancer+stem-like+cells+through+paracrine+FGF/Tbx3+signaling.">Estrogen expands breast cancer stem-like cells through paracrine FGF/Tbx3 signaling.</a> Proc. Natl. Acad. Sci. U.S.A. 14 (50), 21737-21737 (2010).</li><li>Bhardwaj, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+protein+phosphatase+2A+activity+alters+androgen-independent+growth+of+prostate+cancer+cells:+therapeutic+implications.">Modulation of protein phosphatase 2A activity alters androgen-independent growth of prostate cancer cells: therapeutic implications.</a> Mol. Cancer. Ther. 10 (5), 720-731 (2011).</li><li>Cheng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AKT2,+a+putative+oncogene+encoding+a+member+of+a+subfamily+of+protein-serine/threonine+kinases,+is+amplified+in+human+ovarian+carcinomas.">AKT2, a putative oncogene encoding a member of a subfamily of protein-serine/threonine kinases, is amplified in human ovarian carcinomas.</a> Proc. Natl. Acad. Sci. U.S.A. 89 (19), 9267-9267 (1992).</li><li>Fernandez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akt1+and+Akt2:+differentiating+the+aktion.">Akt1 and Akt2: differentiating the aktion.</a> Histol. Histopathol. 26 (5), 651-651 (2011).</li><li>Luo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+RNAi+screen+identifies+multiple+synthetic+lethal+interactions+with+the+Ras+oncogene.">A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene.</a> Cell. 137 (5), 835-835 (2009).</li><li>Ketela, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+platform+for+highly+multiplexed+mammalian+functional+genetic+screens.">A comprehensive platform for highly multiplexed mammalian functional genetic screens.</a> B.M.C. Genomics. 12 (1), 213-213 (2011).</li></ol>

Matthew J. Coussens, Courtney Corman, Ashley L. Fischer, Jack Sago, and John Swarthout are employed by Sigma-Aldrich, which makes tools and reagents used in this article.

In this study, we present a genome-wide screen to identify genes involved in cytotoxicity of A549 cells following paclitaxel/TRAIL treatment. The use of a pooled approach in a genome-wide screen reduces the time, cost, and equipment investment normally associated with conventional arrayed screens. Critical to the success of the screen are optimization experiments performed beforehand. Transduction conditions and MOI must be empirically determined. 
The selection method should allow for robust selection of cells displaying the desired phenotype (e.g., survival, surface protein expression, etc). Optimization of these parameters will reduce the number of false positives detected.
Here, gDNA was harvested from the bulk population of selected cells and then TOPO cloned to identify individual shRNA inserts. One possible improvement for this experiment would have been to select for live cells using FACS to ensure that only live cells are collected. The identified targets will be validated by transduction with target-specific shRNAs in a screening assay. True hits will display the phenotype in a majority of wells.
The LentiPlex Pooled Library is flexible in its application. It is compatible with a wide array of downstream applications and may be used with either positive or negative selection strategies. Depending on the screening conditions, deconvolution of the shRNA inserts is accomplished via PCR/cloning, microarray, or next-generation sequencing 9, 10.While the power and speed of these techniques to deconvolute RNAi screens is well established, one concern with microarray and next-generation sequencing methods is the availability of bioinformatics resources and knowledge needed to correctly analyze and interpret experimental results. The costs associated with these techniques can often times be a barrier to the undertaking of genome wide screens. A PCR/cloning based approach, while not as powerful as microarrays and next-generation sequencing is an effective and lower cost alternative. 
Agilent now offers a custom array based on the TRC shRNA library to further aid in LentiPlex screens. These options allow researchers to use LentiPlex to study a diverse range of cellular functions.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=SHPH01%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC&cm_mmc=affiliate-_-JOVE-_-video-_-SHPH01" target="_blank">LentiPlex Pooled Array</a></td>
			<td><a href="http://www.sigmaaldrich.com/united-states.html" target="_blank">Sigma-Aldrich</a></td>
			<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=SHPH01%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC&cm_mmc=affiliate-_-JOVE-_-video-_-SHPH01" target="_blank">SHPH01</a></td>
		</tr>
		<tr>
			<td>A549 Cells</td>
			<td>ATCC</td>
			<td>CCL-185</td>
		</tr>
		<tr>
			<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=T5694%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC&cm_mmc=affiliate-_-JOVE-_-video-_-T5694" target="_blank">TRAIL</a></td>
			<td><a href="http://www.sigmaaldrich.com/united-states.html" target="_blank">Sigma-Aldrich</a></td>
			<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=T5694%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC&cm_mmc=affiliate-_-JOVE-_-video-_-T5694" target="_blank">T5694</a></td>
		</tr>
		<tr>
			<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=T7402%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC&cm_mmc=affiliate-_-JOVE-_-video-_-T7402" target="_blank">Paclitaxel</a></td>
			<td><a href="http://www.sigmaaldrich.com/united-states.html" target="_blank">Sigma-Aldrich</a></td>
			<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=T7402%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC&cm_mmc=affiliate-_-JOVE-_-video-_-T7402" target="_blank">T7402</a></td>
		</tr>
		<tr>
			<td>Topo TA Cloning Kit</td>
			<td>Invitrogen</td>
			<td>K4575-01</td>
		</tr>
		<tr>
			<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=P2893%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC&cm_mmc=affiliate-_-JOVE-_-video-_-P2893" target="_blank">JumpStart Taq ReadyMix</a></td>
			<td><a href="http://www.sigmaaldrich.com/united-states.html" target="_blank">Sigma-Aldrich</a></td>
			<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=P2893%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC&cm_mmc=affiliate-_-JOVE-_-video-_-P2893" target="_blank">P2893</a></td>
		</tr>
		<tr>
			<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=PLN70%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC&cm_mmc=affiliate-_-JOVE-_-video-_-PLN70" target="_blank">GenElute Plasmid Miniprep kit</a></td>
			<td><a href="http://www.sigmaaldrich.com/united-states.html" target="_blank">Sigma-Aldrich</a></td>
			<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=PLN70%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC&cm_mmc=affiliate-_-JOVE-_-video-_-PLN70" target="_blank">PLN70</a></td>
		</tr>
		<tr>
			<td>EcoR1</td>
			<td>New England Biolabs</td>
			<td>R0101</td>
		</tr>
		<tr>
			<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=H9268%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC&cm_mmc=affiliate-_-JOVE-_-video-_-H9268" target="_blank">hexadimethrine bromide</a></td>
			<td><a href="http://www.sigmaaldrich.com/united-states.html" target="_blank">Sigma-Aldrich</a></td>
			<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=H9268%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC&cm_mmc=affiliate-_-JOVE-_-video-_-H9268" target="_blank">H9268</a></td>
		</tr>
	</tbody>
</table>

mission lentiplex pooled shrna library screening in mammalian cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.
There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids 1. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.
The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes 2. Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 108 TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.
Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line 3, 4. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.

Watch this Scientific Journal Video about MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells at JoVE.com

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to ...

mission-lentiplex-pooled-shrna-library-screening-in-mammalian-cells

Research

JoVE Journal

Biology

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...