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Wellcome Trust Sanger Institute Team Publishes Details on Mouse miRNA Knockout Resource

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By Doug Macron

A team of researchers from the Wellcome Trust Sanger Institute this week published a report in Nature Biotechnology detailing the creation of mirKO, a library of mouse embryonic stem-cell clones with deletions for the “majority” of microRNAs in the miRBase miRNA database.

According to Haydn Prosser, lead author on the paper, the library is the result of a three-and-a-half-year effort and is first of its kind available to the general research community — the only other genome-wide miRNA knockout resource available is for C. elegans. It is expected to facilitate investigations into the role of the non-coding RNAs, both individually and within clusters.

The dysregulation of miRNA expression has been linked to a number of diseases including cancer, and “the role of miRNAs in cellular proliferation and differentiation has raised opportunities for new strategies for therapeutic intervention in the disease process,” the investigators wrote in their paper.

And though coding gene knockout resources exist for the mouse, “much of our knowledge of miRNA function in mice comes from over-expression studies or the disruption of all miRNAs by tissue-restricted knockout of the processing enzyme Dicer,” they wrote. “Rigorous analysis of miRNA function will require the creation of compound mutants of miRNA family members.”

To address this need, the team created mirKO with the goal of providing a “research toolbox of mutant alleles that can be used as reagents for studying miRNA function in a range of complementary approaches,” according to the paper.

The researchers began by designing targeting vectors that would generate small deletions for individual miRNAs. For cases where miRNA genes were close together, the vectors were designed to delete clusters “to minimize the adventitious disruption of their neighbors,” the report's authors wrote.

They set out to make vectors for all of the mouse genes in miRBase except for those on a particular stretch of chromosome 2, since “this region contains several family members of repetitive miRNA genes of low sequence complexity.

“Excluding this region, the 513 other miRNA genes in miRBase corresponded to 455 individual targeting vector designs,” they noted. “Of these, we did not attempt to design vectors for nine, and a further eight failed at the automated vector design stage, leaving 438 possible vector designs that correspond to … 97 percent of the miRNA genes on our target list.”

To generate the library, the researchers used ES cells from the C57BL/6N mouse strain, the standard genetic background for knockouts, “enabling the mutant alleles to be produced in a defined genetic background and thus avoiding the complication of assessing mouse phenotypes in an outbred population,” they noted.

Seventy percent of targeting vectors yielded at least one targeted clone from the first set of 32 clones analyzed, with an overall average targeting frequency of 25 percent, according to the paper.

“In cases where targeting failed, or generated fewer than three positive clones, we performed additional rounds of targeting and screened successive sets of clones, which increased the overall vector success rate to 81 percent,” the paper's authors wrote. “Overall, we have successfully generated targeted deletions for 392 of the intended 494 miRNA genes, either as singletons or as clusters, after excluding candidate miRNAs that were removed from later versions of miRBase.”

Though configured as simple deletions of the miRNAs, the knockouts are designed to enable “efficient post-targeting modification” through recombinase-mediated cassette exchange, allowing for the production of multiple alternative alleles, they stated.

“Other examples of possible applications might include the variation of miRNA copy number and mutating individual miRNAs within clusters,” they added.

Technical information regarding mirKO has been entered into the International Knockout Mouse Consortium website.

“Contingent upon successful expansion and genotyping quality control by long-range PCR, targeted ES cell clones are being transferred to repositories for distribution,” according to the Sanger team.

To date, repositories at the University of California, Davis, and the Helmholtz Zentrum Munchen have signed on to offer mirKO.


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