Archive for the ‘RNAi Screening’ category

The 2012 Cell Biology Meeting: A Good Finish to the Year

January 4th, 2013

2013 seems to have started at a full sprint. It feels as though the annual meeting of the American Society of Cell Biology in San Francisco just finished. We exhibited and presented a tutorial there entitled, Find Functionally Important Driver Genes with RNAi Genetic Screening
Using Pooled shRNA Libraries on December 17. Now in the first week of January, the holidays seemed to have come and gone with barely a skip in stride.

With almost 7,500 attendees, the Cell Biology meeting was a good finish to the year—a chance to catch up with old colleagues and meet some new ones.
Cellecta exhibit booth at ASCB 2012
With the focus on Cell Biology, of course there was a full complement of microscope and imaging companies. Our exhibit was situated near Zeiss which had a rotating sequence of exceptional images of drosophila larvae, tissues, and butterfly wings. Some we couldn’t figure out and neither, it seemed, could anyone at the Zeiss booth. You can see similar ones from Zeiss here with details on the image subject.

Salt Lake City based Vutara, who specialize in molecular resolution imaging, were located right next to us. A spin off of technology developed at the University of Utah, which seems to have an active incubator program. They told us that the University of Utah produces as many start ups as MIT, which surprised me. Evidently, it’s true though. The school seems to have a pretty effective Technology Commercialization Office.

Of course, the exhibitors represented a broad range of technology. For instance, across from our site, was Chromotek, a small German company who offer unique single-chain Alpaca antibody reagents. They came in just for the conference and were giving away cool flashing yo-yos. Also, similar sounding Cellectis, a French company specializing in targeted gene knockout technology, was just a few rows away. They are a much larger company than I realized with significant R&D efforts in drug development. And, B-Bridge International, who helped us get our start, were exhibiting the mechanical cell stretching instrument they distribute for Japanese manufacturer STREX.

Finally, there was also an interesting initiative from the US Dept. of Health and Human Services to consolidate all the regulatory information concerning Hopefully, this project will help eliminate the time and frustration in searching the websites of various government regulatory agencies to see which rule apply when shipping or working with certain biologics. Here’s more about the S3: Science, Safety, Security project.

All-in-all the conference was a good way to end the year. As we start the new one, we wish all our friends, customers, and colleagues a healthy, happy, and prosperous 2013.

Here’s a copy of the slides from the presentation.

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Posted in Conferences, RNAi Screening

Second Phase SBIR Contract from the National Cancer Institute (NCI) to Identify Lethal Gene Combinations in Cancer Cell Models

October 23rd, 2012

It’s been a while since the last post to this blog. It is not that nothing has been going on, in fact, quite the opposite. It has been quite busy the past several months and, unfortunately, blog postings have suffered. However, I thought I would get things started again with short post about our recent SBIR Contract.

Cellecta has been awarded an SBIR Phase II Contract from the NIH National Cancer Institute to continue its work using RNAi screens to identify paired combinations of DNA damage and repair (DDR) genes essential for cancer cells. Some results from the first phase of the grant were posted previously: http://cellecta.com/blog/2011/08/31/rnai-screen-cancer-synergistic-lethality. That work was essentially a proof of principle targeting combinations of 40 DDR genes. With this continuation of the contract, we can now move into a full scale screen of over 400 different DDR genes and run the screen in multiple cell models so we can identify which gene combinations are essential in each.

The purpose of the screen is to identify synthetic lethal genes–genes that, when inactivated together, have a significantly stronger lethal effect than when either is inactivated alone. The idea is that combination therapies could be developed using drugs that target both of the genes together. This combination approach makes is less likely for cancer cells to develop resistance to treatment.

With array based screening, however, it is extremely resource and labor intensive to identify these lethal combinations. However, a pooled RNAi screening approach using a library that co-expresses pairs of shRNAs targeting all combinations of the DDR genes enables efficient screens of tens of thousands of combinations.

We are making the dual shRNA expression libraries now and looking forward to getting these combinational screens started as soon as possible. As soon as we have something interesting, I’ll post it here—so keep an eye out for updates.
You can read the press release about Cellecta’s new SBIR Contract at http://www.prweb.com/releases/20121023/cellecta-rnai-screening/prweb10043967.htm

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Posted in Cancer, NIH, Pooled shRNA Library, RNAi Screening, Synergistic Lethality, Synthetic Lethality

Another Group Finds Similar Keys to Optimal Pooled shRNA Library Screens

March 20th, 2012

Our group recently ran across an article describing an independent RNAi screen with a non-Cellecta pooled shRNA expression library that piqued our interest. In the October 2011 online Genome Biology Journal, Sims, et al. comprehensively described how to run a rigorous genome-wide pooled RNA interference screen using next generation sequencing. The article thoroughly describes the procedural steps involved in screening a heterogeneous pooled library of thousands of lentiviral shRNA expression constructs. Although they used a library somewhat different than our design (the lack of unique sequenceable barcodes being one notable difference), the study nicely demonstrates many of the requirements to ensure meaningful screening results and emphasizes the need to use high throughput next-generation sequencing (as opposed to microarray hybridization) for reproducible measurements of shRNA depletion or enrichment following selection.

Viability or “drop-out” screens that look for depletion of shRNA sequences in selected populations to identify essential genes are one of the most common applications of pooled shRNA screening. The Sims et al. study focuses primarily on the key factors to ensure reproducible results for these screens. Among the most important ones, they note the following:

The shRNA expression library itself must be generated systematically to minimize variation in hairpin representation. This should be assessed by HT sequencing of the plasmid form of the library. Interestingly, Sims et al. also found that the plasmid library is a better reference for starting hairpin representation than the pseudoviral packaged library, which is consistent with our experience at Cellecta, too.
It is essential to manage cell numbers to maintain hairpin representation through the whole screen. Specifically, Sims et al. recommends maintaining at least 1,000 cells per RNA–which is also the ratio we find optimal as described in an earlier blog post. They also caution against letting cells grow past 70% confluency before replating.
Following selection, it is important to amplify sufficient genomic DNA to ensure a representative population from each cell sample. For their library of 10,000 shRNAs, they used at least 60ug of genomic DNA for pre-sequencing PCR amplification. We too find similar amounts necessary (i.e., for 27,000 shRNA, we us 200 ug/sample).
Biological replicates are a requirement to overcome stochastic noise inherent in the screen. However, replicates should have a high level of reproducibility with R-squared values of 0.9 or better.
The pooled shRNA library must be a reasonable size to enable practical handling of the cell populations, genomic DNA amplification, and biological replicates required for an effective screen. Sims et al used a library with 10,000 shRNA.

As a result of the thorough technique, Sims et al. estimated they were able to identify more than 98% of the hairpins in all replicates. One distinct difference in the Sims et al. library compared with Cellecta’s is the presence of an barcode, that is, a unique readily identifiable sequence separate from the hairpin sequence that can be used to identify the particular shRNA in the expression cassette. Somewhat confusingly, though, Sims et al. used the term “barcode screen” although no barcode is present in their library. Detection of shRNA levels in selected populations was done by sequencing a portion of the shRNA encoding region. From our experience, use of a separate unique barcode optimized for sequence analysis increases sequencing calls and helps improve replicate correlations. Sims et al. did find that the pre-sequencing PCR step introduced a certain amount of noise in the data, which is consistent with amplification variability of shRNA sequences as opposed to short standardized barcodes.

The consistency of the general findings of this independent study with our experience, however, is very encouraging. Using a similar but distinct library, Sims et al. have uncovered many of the same critical requirements for optimal screening with complex shRNA pools as we have. This alignment emphasizes the importance of these procedural details to obtaining meaningful screening results, and it provides additional support for RNAi screening standards of practice that was the topic of the previous post.

Sims et al. also mentioned the development of two open source programs for computational analysis of pooled shRNA screening results–shALIGN and shRNAseq: http://rock.icr.ac.uk/software/shrnaseq.jsp.

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Posted in Article, Pooled shRNA Library, RNAi Screening, shRNA Design, Technical

Tags: article RNAi Screen shRNA Design shRNA Library

The Need for RNAi Screening Standards

February 3rd, 2012

A couple of months ago at the CHI Discovery on Target Conference, Hakim Djaballah, Director of the HTS Core Facility at the Memorial Sloan Kettering Cancer Center, gave a unique and insightful presentation highlighting the challenges RNAi screening to identify lethal loss of function interaction in oncogenic systems.

For the exceptional benefits RNAi offers as a targeted tool to elucidate gene function, it is still a relatively new technology with limitations, potential, and idiosyncrasies that remain somewhat undefined. These features are especially evident when RNAi is adapted for large-scale screening of gene function where small details in the set-up, screening process, quality of the reagents, and types of cells can significantly affect the variation and consistency in the large amount of data generated. By highlighting some disappointing follow-up results from initially exciting high-profile publications, Dr. Djaballah identified a few critical benchmarks for evaluating RNAi screening.

Dr. Djaballah’s group looked at three potentially high value cancer targets identified in independent loss-of-function RNAi screens. In addition to the publications, his group reviewed the primary screening data in more detail to evaluate the procedure and statistical significance of the data. The first screen, published in May 2009 in Cell (Scholl, et al.), identified STK33 as required for KRAS oncogenic activity. Initially, a very exciting discovery, a number of groups pursued STK33 as a potential therapeutic target. However, subsequent groups (Barbie, et al. and Luo, et al.) failed to find the STK33 among the strong hits in similar screens. A recent publication in September 2011 by Babij, et al. in Cancer Research, which also does not see STK33 as a hit in a similar shRNA screen, presents compelling data indicating STK33 is not, in fact, generally essential for survival KRAS-dependant cells. Although, based on recent letters to the editor in Cancer Research, this does not seem to be the end of the discussion between these two groups, the initial excitement of STK33 seems to have been premature at best.

The KRAS synthetic lethal screens by Barbie et al. mentioned above that did not pick-up STK33 as a strong hit their screen did, however, identify another potentially interesting gene—the IκB kinase TBK1—that appeared essential for, but previously unknown to be involved in, KRAS lethality. This target was not found by other groups and has yet to be confirmed, but Dr. Djaballah had some reservations as to whether statistical analysis of the data really supported this as a true “hit” or simply an outlier. Dr. Djaballah also had similar concerns with a recent Nature Letter in Oct. 2011 by Zuber et al that identified Brd4 as an essential gene and possible therapeutic target in acute myeloid leukemia cells. A more detailed review of the data from this screen revealed some issues with the confidence of this hit, as there was significant variability in the CV values and a couple of the shRNAs were enriched by as much as million fold.

Dr. Djaballah’s discussion was clearly not intended to disparage any specific study, but rather to demonstrate the slippery potential of over-interpreting the extensive data produced by such a powerful approach. Based on the amount of resources and effort put into a complex screen, it can be difficult to maintain the reserve required to coldly and rigorously analyze the experimental design and results in a detached manner and properly assess which candidates really meet the criteria for follow up. As a relatively new screening technology without much in the way of standards and defined good practices, it is easy to prematurely “fall in love” with potentially interesting targets that may be just noise in the data. From his analysis, Dr. Djaballah suggests paying particular attention to the following three aspects:

Do the infections at the appropriate MOI and with sufficient cells to assess the effect. Although Dr. Djaballah was primarily talking about arrayed screens (with single shRNA plasmids in wells), the points is also very valid for pooled screening. It is critical to ensure there are each cells containing each shRNA to be assayed to generate reliable reproducible results.
Use correct passage times. Whether the screen requires looking at knockdowns or survival, it is important that the cells are maintained for a long enough passage number to produce a significant differential between affected and non-affected cells. Conversely, too many passages will introduce too much noise.
Pay attention to general data and overall results. It is important to see if the overall screening results make sense. For example, are known lethal genes showing up in the hits?

While pointing out the importance of these considerations in evaluating a screening, Dr. Djaballah contended overall that there are currently few standards of practice to provide guidelines around these and similar procedural details. We at Cellecta would agree, having focused most of our effort in the last several years toward optimizing the many subtleties of pooled shRNA screening to enable consistent, robust, and interpretable results.

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Posted in Article, Cancer, Conferences, Pooled shRNA Library, RNAi Screening, Technical

Tags: article cancer conference drug targets reproducibility RNAi Screen shRNA Library speaker

Tissue Targeting May Offer an Alternative Therapeutic Approach for Difficult-to-Treat Diseases

October 13th, 2011

We recently received a phase II of our SBIR grant Exploiting Synthetic Lethality of Hematopoietic Lineage Cells to Develop Novel Targets from the NIH. Rather than trying to identify potential drug targets in oncogenic hematopoietic cells, much of the effort for this project focuses on trying to develop a pharmacologic approach to identify and kill off all hematopoietic cells (see recent press release). This sort of capability may offer an alternative therapeutic approach relying on tissue ablation and renewal to treat hematopoietic cancers such as leukemia and lymphoma.

Clinical approaches exist to regrow and regenerate portions of many essential tissues. For serious diseases, this capability offers a somewhat aggressive treatment possibility where affected tissues are completely eliminated and replaced by new healthy tissue. Blood is one such tissue where that can be regenerated with current clinical procedures. Though risky, a patient’s blood can be regenerated from a bone marrow cell graft through autologous hematopoietic stem cell transplantation and this is a currently a treatment of last resort for individuals suffering with life-threatening blood or bone marrow cancers. However, there is also much focus on regenerative approaches with other tissues, such as bone and skin. In addition, loss of other tissues such as thymus, prostate, and ovary do not have a significant negative impact on the quality of life. As research advances, it is reasonable to assume regenerative approaches will be available for an increasing range of cell types and tissues.

Since all cells of one tissue or lineage type are removed and replaced using this approach, the specific pathology is not particularly significant for ablative and regenerative treatments. Rather than targeting specific cells based on certain disease biology, eradication of all cells in a particular class eliminates the disorder regardless of its nature. This opens up a real opportunity to develop effective therapies for a range of diseases for which there are currently limited treatment options. As clinical technology develops, stem cell therapies improves, more tissue regeneration protocols are established, and in vitro tissue and organ culture technology becomes routine, ablative/replacement treatments may become the preferred therapeutic approach to treat any number of a broad range of disease states.

A major hurdle with this sort of therapy, however, is the in situ eradication step of the diseased cell or tissue. Currently, the principal ways to eliminate damaged cells types or tissues are through localized excision via surgery or radioactive ablation. For example, with autologous hematopoietic stem cell transplantation mentioned above, much of the toxicity of the treatment is associated with the general full-body radiation treatments to which patients are subjected to ablate an individual’s endogenous bone marrow before grafting in new healthy tissue. More precisely targeted approaches to eliminate affected cells are necessary if tissue replacement is to become a generally useful treatment option. Pharmacologicals that target and kill specific types of cells would provide a much needed solution for this problem, and may be easier to develop than drugs that specifically target only diseased, but not healthy, cells. The first step in developing these sorts of targeted molecules is identifying unique tissue-specific markers and potential drug targets as we are attempting for hematopoietic cells with this project.

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Posted in Cancer, NIH, Pooled shRNA Library, RNAi Screening, Synergistic Lethality, Synthetic Lethality

Tags: cancer disease drug targets lethal leukemia lymphoma NIH RNAi Screen shRNA Library stem cell transplantation synthetic lethality

Screening for Synergistically Lethal Knockdown Combinations in Cancer Cells

August 31st, 2011

Therapeutic approaches using multiple drug combinations have become a standard treatment model for many types of cancer. Due to the tremendous genetic complexity and adaptive nature of most human malignancies, the use of multiple drugs acting on different targets increases the efficacy and helps thwart the development of drug resistance. However, the search for new treatment options and expanding number of drug candidates create a demand for better understanding and prediction of the most effective combinations to expedite evaluation and application in clinical settings.

To help address this challenge, Cellecta responded to an NIH contract request to develop new tools that help assess the effect of combinatorially silencing pairs of genes. We developed a variation of our RNAi pooled screening that systematically identifies and prioritizes gene pairs that, when knocked down, significantly inhibit cancer cell growth. In other words, rather than simply identifying which individual genes are essential for growth of cancer cell lines, we identify which pairs that, when silenced, most significantly inhibit cancer cell proliferation.

In some cases, the loss of two genes may be additive and strongly impair cell growth much more significantly than the loss of either gene independently. In fact, sometimes either gene independently may not have any negative effect on cells but, when both are knocked down, there is a synergistic effect that is very lethal to cells. Conversely, losing the function of two known essential genes may not, in fact, have any more of an adverse affect on cell proliferation than the loss either separately. As a result, then, it is very difficult to predict the effect of a loss of a pair of genes so each combination must be tested.

For this project, we made a specialized lentiviral vector containing two shRNA expression cassettes so the construct expresses two different shRNAs from independent promoters. A library of shRNAs was cloned into each of these shRNA expression cassettes to make a pooled heterogeneous population that expressed all paired combinations of shRNAs. With some cloning tricks, we were able to incorporate a short uniquely identifiable sequence (i.e., a “bar-code”) that identified which two shRNAs were in each vector.

The data below were generated with four shRNAs designed against each of 40 DNA damage and repair genes (160 shRNAs total) so, on completion, there were 25,600 different combinations—160 in the first shRNA position vs. 160 in the second. Using this library, we ran an RNAi lethality screen with an isogenic panel of immortalized human mammary epithelial (HMEC) cells using our standard procedures. We have validated several of the pairs and confirmed the combinatorial effect on cell growth. The approach can be reasonably extended to systematically test all combinations of approximately 200 targets in a single screen.

Cytotoxicity level of shRNA combinations identified in synergistic lethality screen with 27K DDR library in HMEC-TERT cells.

Cytotoxicity (bar-code depletion) level of bispecific shRNA constructs identified in SL screen with 27K DDR library in HMEC-TERT cells. Control – shRNA targeting luciferase gene.

Obviously, this approach provides an alternative to what would otherwise be extremely time consuming and expensive pair-wise individual assays to assess lethal gene knockdown combinations in large numbers of target genes. Moreover, it demonstrates the power and flexibility of pooled library screens to address the challenges of elucidating the multiple functional roles and importance of various genes in the variety of biological model systems used in life science and drug discovery research.

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Posted in Cancer, NIH, Pooled shRNA Library, RNAi Screening, Synergistic Lethality

Tags: cancer combinatorial shRNA library lethal NIH RNAi Screen shRNA Library synergistic lethality

RNAi Screening with An Inducible Promoter: Is There an Advantage?

May 12th, 2011

Inducible expression is often desirable for functional genetic testing to establish clear cause and effect for a specific phenotype. A particular phenotype can be demonstrably linked to expression of a specific cDNA by showing it disappears when transcription is suppressed. Although less direct, similar logic applies to shRNA expression, where a phenotype appears when an shRNA is expressed and disappears when the same shRNA is repressed. To facilitate these types of analyses, we developed inducible versions of both the H1 and U6 RNA polymerase III promoters using the tetracycline repressor element. These promoter constructs were optimized so that the addition of tetracycline (actually, a tetracycline-analog doxycycline) induces expression of the shRNA by inhibiting the tetracycline-element-specific repressor (TetR) from binding and blocking transcription. An example of induced repression of GFP can be seen in the figure below.

Fluorescent cell images indicating inducible shRNA inhibiting GFP expression

We routinely use tetracycline-inducible shRNA promoters to validate the effectiveness of individual shRNA sequences identified in our screenings. In particular, with potentially lethal shRNA that target essential genes for cell viability, it is almost a requirement to prevent expression of the shRNA until the cells are established so that the phenotype of cell arrest, necrosis, or apoptosis can be clearly observed and specifically linked to expression of the shRNA. Since the purpose of a majority of our screens is to identify essential genes required for cell proliferation, these inducible shRNA constructs are essential for validating the identified “hits.”

For general functional shRNA screening, however, inducibility is not always desirable. Although it is often assumed by research groups with whom we interact that a library with an inducible shRNA promoter would produce more reproducible and quantitative hits from a genetic screen, our experience indicates this is not necessarily the case. For example, with viability screens, where we are simply looking for which shRNA sequences inhibit cell proliferation (i.e., shRNAs that are depleted in the overall cell population after several divisions), the need to induce the expression of the library by adding doxycycline to the cells complicates the screening procedure and can introduce some unnecessary variation in the system. Of course, with some screens, it may be preferable or even necessary to use an inducible library. For example, screens to identify genes which repress a particular reporter may be easier to carry out when shRNA expression in the library is repressed until sometime after infection and selection of a baseline population. Also, for in vivo screening, using an inducible library may be almost essential so that significant library shRNA expression and selection does not occur until the cells are established in the mouse model. However, there is no clear benefit to including a defined induction step in any particular screen.

As with most experimental options, choosing between inducible vs. constitutive shRNA expression for a library requires careful consideration of the experimental setup. While the disadvantages are not always so obvious, there are often unforeseen drawbacks in adding seemingly small variables into what is already a technically challenging assay.

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Posted in Pooled shRNA Library, RNAi Screening, Technical

Tags: apoptosis H1 in vivo inducible lethal RNAi Screen shRNA Library U6

Ensuring Comprehensive Screening with Pooled shRNA Expression Libraries

April 3rd, 2011

Researchers are often interested in using a pooled shRNA library for genome-wide RNAi screening to cast a very “wide and unbiased net to identify any and all genes functionally involved in some pathway”. Although it is not difficult to make an shRNA library targeting all human or mouse genes, it is practically very difficult to comprehensively screen using such a library. Careful consideration of starting cell numbers and handling of cells during propagation is essential to ensure thorough screening of pooled shRNA expression libraries, minimize false negatives, and obtain consistent and reproducible results.

First, there is an issue of library complexity since it is necessary to have several shRNAs designed to target each gene. The effectiveness of “validated” shRNA varies from cell-to-cell, and no effective shRNA has been identified for many genes. For these reasons, it is necessary to incorporate several shRNAs for each gene to ensure reasonable knockdown of a high percentage of targets. Cellecta typically designs 5-6 shRNA against each target gene, so more than 25,000 shRNA are required to target 5,000 genes. A library targeting the entire human genome, estimated at just over 23,000 genes, requires approximately 115,000 individual shRNA constructs. While it is not particularly difficult to construct libraries of this complexity, this number of unique shRNA sequences creates technical challenges with representative screening.

Pooled shRNA library screens require quantification of changes in the fraction of each shRNA sequence in selected vs. control cells or starting library. A “hit” occurs when selected cells have significantly more or less of a particular shRNA sequence. Whether one is looking at enrichment of specific shRNA in the selected cells vs. the control (positive selection) or depletion of shRNA in selected cells vs. the control (negative selection), it is critical that the screen begin with sufficient numbers of each shRNA to ensure measured changes in the fraction an shRNA sequence are statistically significant. This means that, if there are very low numbers of specific shRNAs at the start of the screen, small random changes in a drifting population may be difficult to differentiate from significant trends. Simply put, a loss of 2 shRNA is a 20% change if there are only 10 initially vs. 2% if there are 100. For this reason, a least a few hundred cells need to be infected with each shRNA to initiate a good screening. This is demonstrated in the data below where starting with a smaller population of just 50 cells per shRNA (third bar) leads to more variance than starting with a population of 200 cells per shRNA (first bar). This means that starting a screen involves infecting 100 times more cells than the complexity of the library. For a library with 25,000 shRNAs, the starting population should be 2.5 million infected cells, and for a library with 115,000 shRNAs, the starting population should be over 11 million infected cells.

To screen a heterogenous mixture of shRNA expression constructs, however, it is important to have 2-3 times more cells than viral particles to help ensure that most cells are only infected with one shRNA-carrying virus (i.e., a multiplicity of infection [MOI] of 0.3-0.5), so you need to have 2-3 times more cells than the number targeted for infection. Thus, 6-8 million cells are needed to start a screen with libraries of 25,000 shRNAs, and a whole genome library of 115,000 shRNAs would require 25-35 million cells. Since each screen should be done in duplicate, or better, triplicate, the number of cells needed makes a full genome screen with a redundant shRNA library impractical.

Finally, to ensure a comprehensive screen, it is not simply sufficient to start with the right amount of cells. During the screening process, incorrect propagating the cells can completely undercut the representation set up at the initiation of the screen. This is especially true for a negative selection screen, such as a viability screen where one is interested in identifying shRNA that kill or inhibit proliferation of cells, and, therefore, drop out of the population. It is critical to maintain the full library representation that was initially used at the start of the screen. If a portion of propagating cells are removed during propagation (e.g., cells are split), the representation of the library can be skewed in the sample. By doing so, this introduces significant noise. This effect is readily seen in the first two bars of the figure where the effect of starting with sufficient cells (200 cells per shRNA) is completely undercut by splitting cells during propagation so that that the final count of cells after 10 days is the same as the initial number of cells. The correlation between triplicates falls dramatically when the cells are split.

Library representation is often overlooked, especially when the desire is for large-scale unbiased screens. However, without careful consideration in designing screening procedures that reflect the complexity of the library, results of these large-scale screens can produce relatively meaningless data with anecdotal results at best. So, what about genome-wide screening? Our approach with the DECIPHER Project is to provide modules, each targeting approximately 5,000 genes with 27,500 shRNA, which enable comprehensive screening. We are more than half way finished building a series of 5 modules that will target all human genes, and 4 modules targeting all mouse genes. For more information on these libraries, visit the DECIPHER Project website.

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Posted in DECIPHER, Pooled shRNA Library, RNAi Screening, Technical

Tags: DECIPHER reproducibility RNAi Screen shRNA shRNA Library

The DECIPHER Project’s New Human Module Targets 5,000 More Genes

February 24th, 2011

We are excited to have just completed the launch of the 3rd Module of the DECIPHER Project Human Lentiviral RNAi Library. As many of you may know, the DECIPHER Project is an open access platform that provides researchers from academic and non-profit institutions with free pooled shRNA libraries and software for genome-wide RNAi screening. It was established by Cellecta, Inc. in October 2010 under collaboration and joint grants with the Fred Hutchinson Cancer Research Center, the Roswell Park Cancer Institute, and The Scripps Research Institute.

The addition of the 3rd DECIPHER Human shRNA Expression Library Module to the DECIPHER pooled shRNA library collection adds approximately 5,000 new cell surface, extracellular, and DNA binding target genes to the 10,000 well-annotated signal transduction and disease-associated genes targeted by the other two Human shRNA expression library modules that have been available since last October. Combined, all three human modules enable functional screening of over 15,000 expressed transcripts—the majority of annotated human genes. This puts us significantly closer to our goal of providing 5 modules that target the entire genome.

The DECIPHER Project libraries are made to the same standards as all Cellecta libraries. Each individual library module targets approximately 5,000 well-annotated genes with 27,500 shRNAs (~ 5-6 shRNA target each transcript). The shRNA template oligonucleotides used to make the libraries are produced using Agilent’s array-based oligo synthesis platform which ensures relatively even representation of each oligo since each is synthesized on its own “spot” in situ on a glass surface. In addition, each shRNA insert in each of the libraries includes a unique bar-code identifier that enables its accurate identification by HT sequencing. This allows precise quantification of all shRNA species in the library and reliable measurement of shRNA quantities after screening. We essentially measure the frequency of each of the 27,500 shRNA sequences in the library. As a result, we know if any are missing after the cloning and packaging steps, and that greater than 95% of the shRNA sequences in each of our pooled shRNA libraries are present at sufficient levels to ensure statistically accurate results.

More information about Cellecta’s library and screening technology is available from Cellecta (www.cellecta.com) or through the DECIPHER Project website (www.decipherproject.net).

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Posted in DECIPHER, Pooled shRNA Library, RNAi Screening

Tags: DECIPHER FHCRC Fred Hutchinson Cancer Research Center RNAi Screen Roswell Park Cancer Institute RPCI shRNA Library The Scripps Research Institute TSRI

Cellecta’s DECIPHER Project RNAi Screening Tools at Roswell Park Cancer Institute

February 14th, 2011

We have started this blog as a simple, unobtrusive conduit that enables us to talk directly to our customers, collaborators, and other interested researchers about new and interesting developments related to Cellecta’s technology and business. As the first post, we thought we would tell you about our recent agreement with the Roswell Park Cancer Institute (RPCI) that provides support to laboratories in their institution doing genome-wide RNAi screenings using our expanding portfolio of open-access DECIPHER™ pooled lentiviral shRNA expression libraries.

Although the plasmid versions of the DECIPHER shRNA library “modules” are available free-of-charge to any academic laboratory though the DECIPHER Project (www.decipherproject.net), the DECIPHER Technology Access and Maintenance (TAM) Program, which the RPCI just joined, lets them make this screening technology accessible through a well-supported centralized core facility. The DECIPHER TAM Program enables us to provide this institution with new modules of the packaged, ready-to-use shRNA expression libraries as they are developed, proactive support for all the labs using these resources, updates on new technological developments and protocols, and access to the latest software to analyze screening results.

Currently, as part of the open-access DECIPHER Project, there are 4 pooled shRNA library modules freely available—two modules targeting 10,000 human genes and two targeting 10,000 mouse genes. Each individual library module targets approximately 5,000 well-annotated genes with 27,500 shRNAs (5-6 shRNA target each transcript). Human and mouse 1 modules target that same set of genes related to signal transduction and cancer. The second modules target a broader range of genes that appear to be involved in disease processes and pathology but were not targeted in the first module. In a few weeks, we will release a human module 3 targeting 5,000 genes. Ultimately, there will be 5 modules targeting all human genes. As with all of our libraries, we utilize bar-coded inserts and have checked the quality of the DECIPHER modules using HT sequencing to ensure all shRNA sequences are present at sufficient levels to ensure comprehensive screening of the targeted gene set.

The ability to identify genes with specific functional activities makes screening with pooled shRNA expression libraries a very powerful technique to investigate the genetic controls regulating a wide range of biological responses. However, effective screening can be technically challenging. While the DECIPHER Project is helping to fulfill our goal to make basic tools for this type of analysis readily available to all labs, the DECIPHER TAM Program allows us to provide the resources and support that will empower labs to utilize these RNAi screening tools to their maximum potential. We are really looking forward to working with RPCI on this program.

More information about the DECIPHER Project can be found on www.decipherproject.net.

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Posted in Cancer, DECIPHER, Pooled shRNA Library, RNAi Screening, TAM Program

Tags: cancer DECIPHER disease RNAi Screen Roswell Park Cancer Institute RPCI shRNA Library signaling pathways TAM