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In Vivo Gene Silencing by Virally Delivered MicroRNA

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Loss of function is a standard approach to elucidate the function of a specific protein. Among these multiple strategies for silencing genes in living animals, genetic knockout in mice have been so far most frequently used. However, short hairpin RNAs (shRNAs) and microRNAs (miRNAs) delivered into the brain by viruses can achieve region-specific gene knockdown in any species at much lower cost and with shorter turnaround time. Recent advances in understanding of the endogenous miRNA function enabled the design of miRNAs as well as miRNA-adapted shRNA that efficiently enter the miRNA-processing pathway and mediate the gene silencing. Predesigned and premade miRNA for many rodent and human genes are now available commercially. Lentiviral vectors designed to express miRNA along with a fluorescent marker are also widely available. Here, we describe the use of virally delivered miRNAs for gene knockdown in living animals. The technique involves multiple procedures starting from the selection of appropriate miRNA sequences, then preparation of the lentiviral vector, production of the infections lentivirus suitable for the in vivo delivery, injection of the virus into the brain, and the testing of the animals for the measure of interest such as behavior, and, finally, post hoc determination of the infection efficiency and the degree of the in vivo knockdown in each animal.
The virally delivered miRNA knockdown is powerful enough to achieve physiologically relevant protein knockdown in the brain of living animals. Furthermore, the knockdown procedure is flexible enough to be adapted to requirements of almost any in vivo experiment, and, thus, has a large yet unrealized potential.
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