Protocols for Use of Homologous Recombination Gene Targeting to Produce MicroRNA Mutants in Drosophila
MicroRNAs (miRNAs) are noncoding RNA molecules that have come to attract considerable interest for their roles in animal and plant development and disease. One means to study miRNA function in animal development is to create mutations. Use of gene-targeting strategies based on ends-out homologous recombination is a useful approach to produce mutations of desired structure, and is gaining popularity for producing miRNA knockouts. Here we present a detailed protocol for miRNA gene targeting and for their subsequent molecular characterization as well as confirmation by rescue. The descriptions of a series of modified vectors designed to facilitate the analysis of miRNA function are included, and a method to manipulate the mutant genome using recombinase-mediated cassette exchange.
Key wordsMicroRNA Drosophila Gene targeting Homologous recombination φC31 integrase Recombinase-mediated cassette exchange
We thank Rubing Liu, Hai Hwee Tay, Kah Junn Tan, and Yoke Ping Gum for technical support. Susan from Genetic Services Inc. provided injection services ably and with patience when needed. Natascha Bushati and Boris Bryk helped by sharing their experiences in generating miRNA knockouts. We thank Dr. Pernille Rorth for providing hs-Cre strains. This work has been supported by EU-FP6 grant “Sirocco” LSHG-CT-2006-037900, Singapore National Research Foundation under CRP Award No. NRF-CRP3-2008-03, and Temasek Life Sciences Laboratory. Ruifen Weng is a recipient of a Singapore Millennium Foundation Scholarship.
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