This work was supported by The Wellcome Trust, Glaxosmithkline (GSK) and the Scottish Institute for Cell Signalling (now part of the MRC Protein Phosphorylation and Ubiquitylation unit).

1. Assay Development Stage
Note: prior to initiating the siRNA screen, an assay development stage is critical to set out important parameters for screening with the reporter cell line. It is essential to invest significant effort at this stage as this will underpin the future success of the screen.
<ol>
	<li>To characterize the hypoxia-responsiveness of U20S-HRE reporter cell line, grow 2 x 75 cm2 flasks of U20S-HRE cells to 80-90% confluence in Dulbeccos Modified Eagle Medium (DMEM) supplemented with 10% FBS.</li>
	<li>Aspirate the media from one flask of cells, wash twice with 10 ml PBS and detach cells by adding 2 ml 0.05% trypsin-EDTA solution and incubating at 37 &#176;C for 5 min. Resuspend cells in 8 ml DMEM 10% FBS and pipette up and down to create a homogenous cell suspension.</li>
	<li>Pipette 20 &#956;l of the suspension to a cell counting chamber, in duplicate, and insert the chamber into an automated cell counter. Click &#8220;Display Image&#8221; on the cell counter software and ensure the cells are in focus. Click &#8220;Count&#8221; and make a note of the cell density. Repeat this for the other duplicate and calculate the average cell density. Dilute the cells with DMEM 10% FBS to a concentration of 60,000 cells/ml.</li>
	<li>Using a multichannel pipette, add 100 &#956;l (6,000 cells) of the diluted cells to the same three columns (e.g. A5-H5, A6-H6 and A7-H7) of 5 sterile white-walled 96-well assay plates, and transfer to a humidified 37 &#176;C incubator at 5% CO2 overnight. Note: these plates will be used to determine the hypoxia-dependent luciferase output for a range of hypoxia exposures: 0 hr, 2 hr, 6 hr, 10 hr and 24 hr.</li>
	<li>At the end of the following day, note the time and add the 24 hr hypoxia plate to the hypoxia workstation set to 1% oxygen. The following morning, calculate the time 10 hr, 6 hr and 2 hr before the 24 hr plate is due to be removed from the hypoxia workstation. At these times, add the 10 hr, 6 hr and 2 hr plates to the hypoxia workstation.</li>
	<li>Prepare 30 ml of 2x combined luciferase lysis/assay buffer (50 mM Tris Phosphate pH 7.8; 16 mM MgCl2; 2 mM DTT; 2% Triton-X-100; 30% glycerol; 1 mM ATP; 1% BSA; 0.25 mM luciferin and 8 &#956;M Na4P2O7). Note: add the components in the order listed and allow the BSA at least 30 min to dissolve before the addition of luciferin and Na4P2O7.</li>
	<li>Remove all plates from the hypoxia workstation at the appropriate time. Add 100 &#956;l of 2x luciferase lysis/assay buffer to the appropriate wells of the assay plate and cover with clear film. Shake the plates on a plate shaker for 10 min at 500 rpm to thoroughly lyse the cells.</li>
	<li>Transfer the plate stack to an automated 96 well plate luminometer rack. Under the &#8220;Protocols&#8221; menu, select &#8220;abs 595&#8221; and highlight all wells to be read in the on-screen plate map under the &#8220;well selection&#8221; menu. Click &#8220;run&#8221; to measure luminescence of each well then calculate the average reading of each plate. Calculate the hypoxia-dependent fold-increase of reporter activity by dividing the average reading of each hypoxia plate by the average reading of the normoxia (0 hr hypoxia) control plate. Note: it is essential to observe a robust hypoxia-dependent luciferase response to be suitable for screening. A five-ten fold increase in reporter activity is considered excellent.</li>
	<li>Create a master control plate that contains four high control (HIF1A siRNA) replicates (wells A1-D1), four low control (FIH1 siRNA) replicates (wells A12-D12), four buffer only control replicates (wells E1-H1) and four non-target siRNA control replicates (E12-H12), where all siRNA pools are at a concentration of 200 nM. Note: High and low controls are established based on known pathway regulators that increase and decrease the hypoxia response respectively. Alternatively, assay development using a subset of the library may be used to identify suitable controls.</li>
	<li>Using a multichannel pipette, transfer 10&#160;&#956;l of each control siRNA to a sterile white walled assay plate. Prepare transfection mix of 0.1&#160;&#956;l transfection reagent in 10&#160;&#956;l reduced-serum medium (1:100) per well, and transfer 10&#160;&#956;l of transfection mix to each control well. Pipette up and down briefly to mix, and leave the plate to rest for 20-60 min to allow transfection complex formation.</li>
	<li>Prepare a cell suspension of 75,000 cells/ml with the second flask of U20S-HRE cells. Using a multichannel pipette, add 80&#160;&#956;l (6,000 cells) of the cell suspension to each transfection mix. Transfer the plate to a humidified 37 &#176;C incubator with 5% CO2 for 24 hr, then transfer to a hypoxia workstation for a further 24 hr and perform luciferase assays as described in steps 1.6-1.8.</li>
	<li>Calculate the Z-factor for the high and low controls using the formula Z = 1 &#8211; [3 x (standard deviation of high control + standard deviation of low control) / (mean of high control &#8211; mean of low control)]. Note: a value of between 0.5-1 indicates an excellent assay and should be strived for.</li>
</ol>
2. Primary Screen
Note: once these basic conditions from the assay development stage are in place, the primary screen can be performed in triplicate in 96 well-plate format using the following protocol.
<ol>
	<li>Grow 7 x 75 cm2 flasks of U20S-HRE cells to 80-90% confluence in DMEM supplemented with 10% v/v fetal bovine serum (FCS). 
	Note: the following steps 2.2-2.13 should be carried out on Day 1.</li>
	<li>Initiate replicate 1 by preparing a master control plate containing 200&#160;&#956;l of each control as described in step 1.9 and thawing out a dilution series of the siRNA ubiquitome library for a minimum of 30 min. Note: each well contains a pool of 4 siRNAs assembled in 17 x 96-well plates with columns 1 and 12 left empty for controls.</li>
	<li>In a laminar flow hood, label (or bar-code) 17 sterile white walled assay plates with lids from numbers 1-17, corresponding to each plate of the siRNA library series.</li>
	<li>Centrifuge the master control plate and the siRNA ubiquitome library briefly (1 min at 2,000 x g) to ensure all siRNAs accumulate at the bottom of the well.</li>
	<li>Use an automated liquid dispenser to robotically &#8220;stamp&#8221; 10&#160;&#956;l of each control from the master control plate onto the 17 assay plates, using the same 96-well tip stack for each plate.</li>
	<li>Using a fresh 96-well tip stack for each of the 17 library plates, transfer 10&#160;&#956;l of each ubiquitome siRNA to its corresponding assay plate using an automated liquid dispenser.</li>
	<li>Prepare 40 ml transfection reagent for the 17 assay plates using the ratio established in 1.10. Note: this includes an additional 20 ml to account for the &#8220;dead volume&#8221; in the cell dispenser. The &#8220;dead volume&#8221; refers to the volume of liquid continually present within the tubing network in the cell dispenser.</li>
	<li>Use an automated cell dispenser to transfer 10&#160;&#956;l of transfection reagent to each well in the 17 assay plates. Briefly shake the assay plates on a shaker (1 min at 500 rpm) to allow thorough mixing of siRNA and transfection reagent, then leave still at room temperature for 20-60 min to allow siRNA:transfection reagent complex formation.</li>
	<li>Wash two 75 cm2 flasks of U20S-HRE cells twice with 10 ml PBS and detach with 2 ml trypsin-EDTA at 37 &#176;C for 5 min. Add 8 ml DMEM 10% FBS to each flask and pipette up and down several times to create a homogenous cell suspension. Combine cells from both flasks and transfer to a sterile 50 ml plastic tube.</li>
	<li>Calculate cell concentration by pipetting 20&#160;&#956;l cells in duplicate to a cell counting chamber and calculate the average cell concentration from two readings of an automated cell counter as described in step 1.3.</li>
	<li>Prepare 155 ml of cell suspension by diluting with DMEM 10% FBS to a concentration of 75,000 cells per ml in a sterile plastic container. Note: this volume is sufficient for 17 plates and includes an additional 25 ml for the dead volume in the cell dispenser.</li>
	<li>Add a sterile magnetic stirrer to the cell suspension and place on a stirrer set up inside the laminar flow hood to limit cell clumping. Equilibrate the automated cell dispenser with the cell suspension and set it to dispense 80&#160;&#956;l of cells per well (6,000 cells).</li>
	<li>Place each of the 17 plates, in turn, onto the automated cell dispenser and dispense cells. Record the time and stack plates in groups of 5 then place stacks in a humid 37 &#176;C incubator at 5% CO2 for 24 hr. Note: the final concentration of siRNA pool will be 20 nM in 100&#160;&#956;l total volume. 
	Note: the following steps 2.14-2.15 should be carried out on Day 2.</li>
	<li>Initiate the second replicate on day 2, following the procedure outlined in Day 1 for replicate 1 (2.2-2.13).</li>
	<li>After 24 hr of transfection, transfer plates from replicate 1 in a sterile environment to a hypoxia workstation set at 1% oxygen and leave for 24 hr to induce the HRE reporter. 
	Note: the following steps 2.16-2.20 should be carried out on Day 3.</li>
	<li>Initiate the third replicate, following the procedure outlined in Day 1 for replicate 1 (2.2-2.13).</li>
	<li>After 24 hr of transfection, transfer plates from replicate 2 in a sterile environment to a hypoxia workstation set at 1% oxygen and leave for 24 hr to induce the HRE reporter.</li>
	<li>Two hours before replicate 1 plates are to be removed from the hypoxia workstation, prepare 200 ml of 2x luciferase lysis/assay buffer as described in 1.6. Note: this volume includes an additional 20 ml to ensure the buffer reservoir remains well covered.</li>
	<li>Remove replicate 1 assay plates from the hypoxia workstation after 24 hr hypoxia exposure and using an automated liquid dispenser, add 100&#160;&#956;l of 2x luciferase lysis/assay buffer to each assay plate and cover with clear film.</li>
	<li>Shake the plates on a cell shaker for 10 min at 500 rpm to thoroughly lyse the cells. Transfer each plate in turn to a luminometer plate reader and record luminescence as described in step 1.8. 
	Note: the following steps 2.21-2.22 should be carried out on Day 4.</li>
	<li>After 24 hr of transfection, transfer plates from replicate 3 in a sterile environment to a hypoxia workstation set at 1% oxygen and leave for 24 hr to induce the HRE reporter.</li>
	<li>Read the replicate 2 plates by following steps 2.18-2.20 used for replicate 1. 
	Note: the following step 2.23 should be carried out on Day 5.</li>
	<li>Read the replicate 3 plates by following step 2.18-2.20 used for replicate 1.</li>
	<li>Compile all 3 replicates of the primary screen, and calculate the Z factor for each plate using the formula given in 1.12. Note: if any plate has a Z factor of less than 0.5, consider repeating this plate to improve the data quality.</li>
	<li>Calculate the coefficient of variance (CV) for the high and the low controls by using the following formula: 100 x standard deviation of control/ average of control. Note: CV should be as low as possible, it is reasonable to expect 10-15% variation. If variation is significantly higher, consider repeating the plate.</li>
	<li>Calculate percent activation of each siRNA per plate by using the following formula: [(mean of high control &#8211; siRNA score) / (mean of high control &#8211; mean of low control)] x 100. In addition, calculate the non-target (NT)-fold of each siRNA by using the formula [Sample data/mean of NT]. For both percent activation and NT-fold, calculate the average of the three replicates and compile the values.</li>
	<li>Using the raw data, calculate the Spearman&#8217;s Rank Correlation Coefficient (SRCC) to determine how closely the three replicate plates correlate using the formula: 
	<img fo:content-width="1in" src="/files/ftp_upload/51572/51572eq1.jpg" width="120" /> 
	where r = SRCC; d = difference between the two numbers in each pair of ranks and n = number of pairs of data. Note: a good correlation between plate replicates will give values close to 1. If one replicate shows low SRCC against the other 2 plates, investigate further and consider repeating that plate.</li>
	<li>Decide which siRNAs are of sufficient interest for secondary screening (up to 80). Employ user-defined cut offs (e.g.&#160;selecting all siRNAs showing less than 5% or over 95% percent activation, or less than 0.5 NT-fold or over 1.5 NT-fold) or apply bioinformatics or knowledge-based reasoning to search for clusters of hits falling within the same UBL pathway. Note: normally, a combination of these approaches determines which siRNA are taken into secondary screening.</li>
</ol>
3. Secondary Screen
Note: a confirmatory secondary screen is carried out based on a maximum of 80 siRNAs of interest from the primary screen. This number can be conveniently carried out with each replicate plated on a single 96 well plate with full controls as per the primary screen (see 2.2). It is very useful to confirm that the regulators identified in the primary screen reproducibly elicit the same phenotype, and this will assist in refining the triage decisions on which hits should be carried through to the final tertiary/deconvolution screen.
<ol>
	<li>Grow 1 x 75 cm2 flask of U20S-HRE cells to 80-90% confluence in DMEM with 10% FBS.</li>
	<li>Note the plate number and location of the primary screen hits, and plan their new location on the secondary screen cherry picked master plate, leaving columns 1 and 12 free for controls. Prepare the control master plate as in 1.9 but with a total volume of each control of 50 &#956;l.</li>
	<li>Thaw a second dilution series of the ubiquitome library (set aside for cherry-picking individual siRNA pools) for a minimum of 30 min. Centrifuge the plates briefly (1 min at 2,000 x g) then add 50&#160;&#956;l of the secondary screen siRNAs to their new plate locations on the cherry picked master plate.</li>
	<li>Carry out the secondary screen using the same basic protocol outlined for the primary screen with the following modifications: (a) run all three replicates on the same day in one assay plate per replicate and (b) adjust volumes of reagents accordingly.</li>
</ol>
4. Tertiary/Deconvolution Screen
Note: tertiary or deconvolution screening is performed on a maximum of 20 siRNAs from the secondary screen. This step is to examine the effect of knockdown using each individual siRNA from the original pool of four. Normally, at least two individual siRNA duplexes from each pool should illicit the same phenotype to have a reasonable degree of confidence that the observed phenotype is not due to siRNA off-target effects. To further increase confidence at this stage, additional individual siRNA duplexes targeting the gene in question can be designed and tested for their ability to elicit the given phenotype. Results from these experiments may then be used to calculate the H score, where H = 0.6 or over (i.e. where at least 3 out of 5 individual siRNAs elicit the phenotype) is considered acceptable6.
<ol>
	<li>Grow one 75 cm2 flask of U20S-HRE cells to 80-90% confluence in DMEM with 10% FBS.</li>
	<li>Create a plate map for the tertiary screen by planning the location of the four individual siRNA duplexes of each hit on the tertiary screen master plate, leaving columns 1 and 12 free for controls. Prepare the control master plate as in 1.9 but with a total volume of each control of 50 &#956;l.</li>
	<li>Thaw the deconvolution/individual siRNA plates and add 50&#160;&#956;l of the individual siRNAs (200 nM) into each well of the tertiary screen master plate according to the plate map (4.2) to allow for three x 10&#160;&#956;l replicates and a comfortable 20&#160;&#956;l excess.</li>
	<li>Carry out the tertiary screen using the same basic protocol outlined for the primary screen with the following modifications: (a) run all three replicates on the same day in one assay plate per replicate and (b) adjust volumes of reagents accordingly</li>
</ol>
Note: Ideally the threshold for individual siRNA duplexes should be set at the same stringency cut-off as for the pool. However, it may be acceptable to relax the threshold by 10-20% for individual siRNAs, especially where at least one other duplex falls within the set threshold. It is worthwhile bearing in mind that the individual effect of siRNAs may be less than that of the pool, and conversely, in some cases individual siRNAs may show a stronger effect on the cellular phenotype in isolation than when it exists in the pool (even when concentration is accounted for).

<ol>
	<li>Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+function+of+ubiquitin-like+proteins.">Origin and function of ubiquitin-like proteins.</a> Nature. 458, 422-429 (2009).</li><li>Schulman, B. A., Harper, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin-like+protein+activation+by+E1+enzymes:+the+apex+for+downstream+signalling+pathways.">Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways.</a> Nat Rev Mol Cell Biol. 10, 319-331 (2009).</li><li>Siomi, H., Siomi, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+road+to+reading+the+RNA-interference+code.">On the road to reading the RNA-interference code.</a> Nature. 457, 396-404 (2009).</li><li>Kaelin, W. G., Ratcliffe, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+sensing+by+metazoans:+the+central+role+of+the+HIF+hydroxylase+pathway.">Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway.</a> Mol Cell. 30, 393-402 (2008).</li><li>Melvin, A., Mudie, S., Rocha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chromatin+remodeler+ISWI+regulates+the+cellular+response+to+hypoxia:+role+of+FIH.">The chromatin remodeler ISWI regulates the cellular response to hypoxia: role of FIH.</a> Mol Biol Cell. 22, 4171-4181 (2011).</li><li>Bhinder, B., Djaballah, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+analyzing+actives+in+random+RNAi+screens:+introducing+the+&amp;#34;H+Score&amp;#34;+for+hit+nomination+&amp;+gene+prioritization.">A simple method for analyzing actives in random RNAi screens: introducing the &amp;#34;H Score&amp;#34; for hit nomination &amp; gene prioritization.</a> Comb Chem High Throughput Screen. 15, 686-704 (2012).</li><li>Bett, J. S., 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=The+P-body+component+USP52/PAN2+is+a+novel+regulator+of+HIF1A+mRNA+stability.">The P-body component USP52/PAN2 is a novel regulator of HIF1A mRNA stability.</a> Biochem J. 451, 185-194 (2013).</li><li>Zhang, J. H., Chung, T. D., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> J Biomol Screen. 4, 67-73 (1999).</li><li>Birmingham, 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=Statistical+methods+for+analysis+of+high-throughput+RNA+interference+screens.">Statistical methods for analysis of high-throughput RNA interference screens.</a> Nat Methods. 6, 569-575 (2009).</li><li>Stagg, H. R., 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=The+TRC8+E3+ligase+ubiquitinates+MHC+class+I+molecules+before+dislocation+from+the+ER.">The TRC8 E3 ligase ubiquitinates MHC class I molecules before dislocation from the ER.</a> J Cell Biol. 186, 685-692 (2009).</li><li>Zhang, Y., 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=RNF146+is+a+poly(ADP-ribose)-directed+E3+ligase+that+regulates+axin+degradation+and+Wnt+signalling.">RNF146 is a poly(ADP-ribose)-directed E3 ligase that regulates axin degradation and Wnt signalling.</a> Nat Cell Biol. 13, 623-629 (2011).</li><li>Jackson, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+profiling+reveals+off-target+gene+regulation+by+RNAi.">Expression profiling reveals off-target gene regulation by RNAi.</a> Nat Biotechnol. 21, 635-637 (2003).</li><li>Birmingham, 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=3'+UTR+seed+matches,+but+not+overall+identity,+are+associated+with+RNAi+off-targets.">3' UTR seed matches, but not overall identity, are associated with RNAi off-targets.</a> Nat Methods. 3, 199-204 (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whither+RNAi.">Whither RNAi.</a> Nat Cell Biol. 5, 489-490 (2003).</li></ol>

The authors have nothing to disclose.

The use of genome-wide siRNA screens in mammalian cells has proven to be extremely valuable in identifying novel regulators of distinct biological pathways. Here, we have described the use of a targeted ubiquitome siRNA screen to identify regulators of the HIF1A-mediated cellular response to hypoxia.&#160; Targeted screens are becoming increasingly attractive as they are generally cheaper, quicker, easier to manage and report only on the pathway components in which the investigators are interested 7, 10, 11.</...

Modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) represents an expansive biochemical system that regulates diverse biological pathways and stress responses.&#160; The covalent attachment of UBLs to their target proteins can have various outcomes regulating the stability, localization, function or interactome of the substrate1.&#160; The enzymatic steps underlying UBL modification were first established for ubiquitin, and now serve as a paradigm for modification with most UBLs, including SUMO, NEDD8, ISG15 and FAT10.&#160; For modification to occur, the carboxylate group of the UBL diglycine motif is first activated by an E1 activating enzyme to form a high-energy thiol that is transferred to the active-site cysteine of an E2 conjugating enzyme.&#160; The E2 then interacts with a substrate-bound E3 ligase to mediate transfer of the UBL onto (usually) a target lysine residue creating a branched chain (isopeptide) linkage2.&#160; Successive rounds of modification can occur to build isopeptide chains onto the substrate, which for ubiquitin can occur through any of its seven lysines, or through its N-terminal methionine to create linear ubiquitin chains.&#160; These modifications form discrete topologies with diverse purposes such as creating new interaction motifs and targeting proteins for degradation prior to UBL removal by specialist proteases.&#160; In the case of ubiquitin there are two E1 enzymes, 30-40 E2 conjugating enzymes, at least 600 E3 ligases and approximately 100 deubiquitylating enzymes (DUBs).&#160; While the pathways are less expansive for the other 10 or so UBLs, the overall ubiquitome complexity affords huge diversity in the biological outcome of a particular UBL modification. &#160;However, while major advances in UBL biology have been made, the precise cellular roles of the majority of these ubiquitome components remain unknown.
The use of short interfering ribonucleic acid (siRNAs) has emerged as a powerful tool in reverse genetics due to the ability of siRNAs to specifically target cellular mRNAs for destruction, allowing the role of individual genes to be examined in different biological contexts3. Whole genome screens have been used to identify and validate new regulators of many cellular processes, and have created a wealth of useful data accessible to the wider scientific community.&#160; However, while whole genome screens have proven extremely useful, targeted screens are becoming increasingly popular as they are cheaper, faster, involve less data management and report only on members of the genome in which the investigator is most interested.&#160; Therefore, to better understand which cellular processes UBL family components are involved in, we have compiled a human siRNA library targeting all known and predicted components of the ubiquitome.&#160; This includes the UBLs, E1 activating enzymes, E2 conjugating enzymes, E3 ligases, ubiquitin-binding domain (UBD)-containing proteins and DUBs.&#160; This library can be used to screen against a wide range of reporter cell lines of distinct biological problems, thus allowing the unbiased identification of novel UBL components governing these pathways.
The following protocol describes how to perform a rigorous targeted siRNA ubiquitome screen to identify novel regulators of the HIF1A-dependent response to hypoxia.&#160; Under normal oxygen tension, HIF1A is subject to prolyl hydroxylation that causes it to be recognized and targeted for degradation by the Von Hippel Lindau (VHL) E3 ligase complex4.&#160; Hypoxia inhibits prolyl hydroxylation leading to the stabilization of HIF1A and its subsequent binding to hypoxia response elements (HREs) to drive gene expression. Here, we describe a screen using U20S osteosarcoma cells stably expressing firefly luciferase under the control of three tandem copies of the Hypoxia Response Element (U20S-HRE cells)5. &#160;This protocol can be adapted for any biological pathway if a robust read-out of reporter activity is achievable and can be coupled with appropriate positive and negative controls.

<table border="0" cellpadding="0" cellspacing="0" width="790">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:288px;">Automated Liquid Dispenser</td>
			<td style="width:147px;">Fluid-X</td>
			<td style="width:200px;">XPP-721</td>
			<td style="width:156px;">http://www.fluidx.eu/BIOTRACK/xpp-721-liquid-handling-system.html</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Automated cell counter</td>
			<td>Nexcelom Bioscience</td>
			<td>Cellometer Auto T4</td>
			<td>http://www.nexcelom.com/Cellometer-Auto-T4/index.html</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hypoxia Workstation</td>
			<td>Ruskin</td>
			<td>In vivo2 300</td>
			<td>http://www.ruskinn.com/products/invivo2300</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Automated Cell Dispenser</td>
			<td>Thermo Scientific</td>
			<td>Matrix Wellmate</td>
			<td>http://www.matrixtechcorp.com/automated/pipetting.aspx?id=11</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Plate Shaker</td>
			<td>Heidolph</td>
			<td>Titramax 1000</td>
			<td>http://www.heidolph-instruments.co.uk/products/shakers-mixers/platform-shakers/vibrating/titramax/titramax-1000/</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Luminometer</td>
			<td>Perkin Elmer</td>
			<td>Envision 2104 Multilabel Reader</td>
			<td>http://www.perkinelmer.co.uk/Catalog/Family/ID/EnVision%20Multilabel%20Plate%20Readers</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">White Walled Assay Plate</td>
			<td>Greiner Bio One</td>
			<td>655083</td>
			<td>http://www.greinerbioone.com/en/row/articles/catalogue/article/37_11/13221/</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Clear Plate Film</td>
			<td>Perkin Elmer</td>
			<td>1450-461</td>
			<td>http://www.perkinelmer.co.uk/Catalog/Product/ID/1450-461</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Name of the Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">siRNA library</td>
			<td>Thermo Scientific</td>
			<td>On-Target Plus</td>
			<td>http://www.thermoscientificbio.com/rnai-and-custom-rna-synthesis/sirna/on-targetplus-sirna/search-gene/</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Transfection reagent</td>
			<td>Invitrogen</td>
			<td>Lipofectamine RNAimax</td>
			<td>http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Protein-Expression-and-Analysis/Transfection-Selection/lipofectamine-rnaimx.html</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Reduced Serum Medium</td>
			<td>Invitrogen</td>
			<td>Optimem</td>
			<td>http://products.invitrogen.com/ivgn/product/31985062?ICID=search-product</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DMEM</td>
			<td>Invitrogen</td>
			<td>41965-039</td>
			<td>http://products.invitrogen.com/ivgn/product/41965039#</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">FBS</td>
			<td>Invitrogen</td>
			<td>16000-044</td>
			<td>https://products.invitrogen.com/ivgn/product/16000044?ICID=search-product#</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tryspin-EDTA</td>
			<td>Invitrogen</td>
			<td>25300-054</td>
			<td>https://products.invitrogen.com/ivgn/product/25300054?ICID=search-product#</td>
		</tr>
	</tbody>
</table>

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.

Prior to screening, the hypoxia-responsiveness of U20S-HRE cells is established.&#160; U20S-HRE cells express a reporter construct consisting of firefly luciferase fused downstream of three tandem copies of the hypoxia response element, which is bound by the HIF1A/HIF1B heterodimer upon exposure to hypoxia (Figure 1A).&#160; Cells are placed in a hypoxia workstation for a range of times to establish which hypoxia exposure produces the most effective response for screening.&#160; U20S cells express low le...

Watch this Scientific Journal Video about siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells at JoVE.com

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

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 ...

sirna-screening-to-identify-ubiquitin-ubiquitin-like-system

Research

JoVE Journal

Biology

11.3K visualizações.  University of Dundee.  Aqui, descrevemos uma metodologia para realizar uma siRNA alvo "ubiquitome" tela para identificar romance ubiquitina e reguladores de ubiquitina-like da resposta celular mediada por HIF1A à hipóxia. Isto pode ser adaptado para qualquer via biológica, onde uma leitura robusta de actividade repórter está disponível. 

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

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

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 ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

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.

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 ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

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, ...