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

February 24th, 2011 by pauld-cellecta No comments »
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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

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U6 or H1—Which promoter is better?

February 18th, 2011 by pauld-cellecta No comments »
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Since we provide both constitutive and tetracycline-inducible versions of both U6 and H1 shRNA promoters, we often get questions regarding which to choose or which is “better.” In fact, our data with both promoters indicates they function equally well, so it is difficult to provide a clear-cut answer. We use the U6 promoter primarily for shRNA expression libraries as this is generally thought to be a stronger promoter in most cells. We have not systematically tested this in-house, but it is consistent with our findings where U6-driven constructs provide knockdown in a wide range of cell lines. However, some of our clients have specifically requested H1 constructs because U6 constructs appeared detrimental to the growth of some cell lines, possibly because expression levels are too high. The particular concern mainly seems to be with using U6 in neuronal cells. Just to emphasize, we have not seen this in our research. Conversely, we have not had problems with H1-driven shRNA constructs. In a wide range of cells, it seems to function as well as U6.

Overall, we don’t have a strong recommendation regarding the use of either promoter. It may be that one or the other is better in certain cells or under certain situations. However, in over 50 cell lines that we have worked with, we haven’t seen a clear distinction. We do suggest the researcher try both in their cell systems to check first-hand if one is preferable over the other.

We would be interested to hear from anyone who has had experience with U6 and H1 promoters and what their results were. Comments can be posted in the form below.

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Posted in Lentiviral Vectors

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

February 14th, 2011 by pauld-cellecta No comments »
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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

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