By Doug Macron
Researchers from the Broad Institute-led RNAi Consortium this week published details about a newly developed library of human open reading frames as they continue to expand beyond gene silencing into developing resources for gain-of-function experiments.
In a paper appearing this week in the online edition of Nature Methods, consortium members and collaborators describe how they produced a sequence-confirmed clonal collection of more than 16,000 human ORFs encoded in a novel vector system.
Using this collection, “we created a genome-scale expression collection in a lentiviral vector, thereby enabling both targeted experiments and high-throughput screens in diverse cell types,” they wrote.
TRC was established by the Broad in 2004 as a public/private consortium of RNAi researchers focused on developing and publicly releasing genome-scale sets of virally expressed shRNAs targeting mouse and human genes (GSN 4/9/2004).
In its first, three-year phase the initiative created a library of about 160,000 shRNAs against roughly 32,000 human and mouse genes, and developed methods to use the library for loss-of-function genetic screens.
A second, four-year phase was launched in 2007 to expand that library, increasing the average number of shRNAs per gene from five to eight, but also boosting the overall number of genes targeted. The consortium also developed improved methods for applying the library for screening experiments.
As reported by Gene Silencing News last summer, TRC was preparing for a third phase of work, and although its exact goals had not yet been defined, the consortium did decide that it would expand its focus beyond RNAi to include non-coding RNAs and ORFs.
At the Broad, "we've been working on [open reading frame] libraries outside of the RNAi Consortium, but we want to make that part of the [consortium's] efforts," TRC Project Leader David Root said at the time.
"Complementary to suppressing transcripts is to over-express them," he noted. "People have been doing that for some time, but I think there is still a lot of room to improve the resources for gain-of-function cell-based screening using over-expression constructs. We are working on that and will make it part of phase three."
With the publication in Nature Methods, the consortium has taken a step forward toward meeting that objective.
“Genome-scale RNA interference reagents have recently been created to enable systematic loss-of-function mammalian genomics,” the paper's authors wrote. However, “to perform complementary gain-of-function studies, comparable libraries of arrayed cDNAs or open reading frames are required along with efficient methods to use these reagents in cell-based assays.”
Although genome-scale ORF collections exist and have proven useful as templates for subcloning or recombinatorial transfer between vectors and protein production, there are limits to their “direct applicability,” according to the paper. “The collections vary dramatically in terms of gene representation, format and functionality, as well as quality measures such as the extent of clonality, sequence annotation, and experimental validation.”
“Tellingly, gain-of-function screening now lags behind the use of RNA interference,” it added.
To address this issue, TRC researchers and collaborators created and characterized two publicly available genome-scale human ORF collections: the human ORFeome version 8.1 Entry clone collection, and the Center for Cancer Systems Biology-Broad lentiviral expression library.
Together, the collections comprise 16,172 distinct ORFs mapping to 13,833 genes, with each ORF plasmid derived from a single bacterial colony. Nearly all clones are fully sequenced, the paper states.
Additionally, the collections enable cell-based functional screens because the clones are encoded in a lentiviral expression vector that produces consistent titers and gene expression, and permit delivery to “most cell types.”
The collections were assembled in four phases, the first of which included an expansion of previous collections to 19,281 ORFs in polyclonal format largely using existing protocols, according to the paper. Clonal plasmid isolates were then derived from single bacterial colonies and sequenced, with the resultant data being used to chose clones for inclusion in hORFeome V8.1. Lastly, the clones were transferred to a lentiviral expression vector to create the CCSB-Broad lentiviral expression library.
“To estimate the accuracy of the final collection of expression vectors, we end-read sequenced 325 colonies and confirmed 98.2 percent accurate transfers,” the paper's authors wrote. “The utility of this resource for systematic functional genomic screens in mammalian cells is illustrated by recent results from a screen of a pilot subset
of this collection, which identified new mediators of resistance to Raf inhibition in melanoma.
“Additional pilot experiments confirmed that this resource enables other readouts including immunofluorescence,” they added.
Overall, the TRC team created what they call the “most fully sequenced, flexible, and annotated version of the human ORFeome to date.”
They noted that the entire collection, comprising both source and lentivirus vector-expression clones, is freely available through the ORFeome Collaboration, an alliance between industry and academia founded to provide unrestricted access to fully sequence-validated full-ORF human cDNA clones.
“We anticipate that these collections will greatly facilitate the systematic functional assessment of human genes that mediate cellular phenotypes,” they concluded.
In addition to the Broad, researchers from the Dana-Farber Cancer Institute and Harvard Medical School contributed to the research appearing in Nature Methods.
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