New technique allows generation of mice in which any miRNA can be deleted or replaced with a different allele.
Wellcome Trust Sanger Institute scientists have released details of a new resource comprising a collection of mouse embryonic stem (ES) cell lines engineered with targeted miRNA deletions. The resource includes a collection of 428 miRNA targeting vectors covering 476 of the miRNA genes annotated in the miRBase registry.
These vectors have been used to generate a library of germline-transmissible C57BL/6N mouse ES cell clones harboring targeted deletions for 392 of the miRNA genes. For most of these targeted clones, chimerism and germline transmission can be scored in resulting mice through a coat color marker.
The knockouts have been configured as simple deletions of the miRNAs, but the researchers have developed a technique known as recombinase-mediated cassette exchange (RMCE), which means each of the target miRNA loci can instead be replaced with a different allelic variant, such as a reporter or conditional allele.
Allan Bradley, Ph.D., Hayden Prosser and colleagues report on construction of the new toolkit and the RMCE technology, in Nature Biotechnology, in a paper titled “A resource of vectors and ES cells for targeted deletion of microRNAs in mice.”
Technical details on the library and information on the availability of mirKO cells and reagents through participating public repositories have been posted on the International Knockout Mouse Consortium website.
“The biology of miRNAs will be revealed only when we can rigorously examine their activity, their role in individual tissues, and at specific times in development,” comments professor Bradley, who is director emeritus of the Sanger Institute. “Our paper shows that the tools within mirKO can do that. We have tagged genes with a color reporter, developed a mutation that can be induced when required, and produced mice carrying mutations. This is an important proof of principle.”