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Gene Silencing In Vitro and In Vivo Using Intronic MicroRNAs

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 936)

Abstract

MicroRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular messenger RNAs (mRNAs) that contain either complete or partial complementarity, are useful for the design of new therapies against cancer polymorphism and viral mutation. Numerous miRNAs have been reported to induce RNA interference (RNAi), a posttranscriptional gene-silencing mechanism. Recent evidence also indicates that they are involved in the transcriptional regulation of genome activities. They were first discovered in Caenorhabditis elegans as native RNA fragments that modulate a wide range of genetic regulatory pathways during embryonic development and are now recognized as small gene silencers transcribed from the noncoding regions of a genome. In humans, nearly 97% of the genome is noncoding DNA, which varies from one individual to another, and changes in these sequences are frequently noted to manifest in clinical and circumstantial malfunction; for example, type 2 myotonic dystrophy and fragile X syndrome were found to be associated with miRNAs derived from introns. Intronic miRNA is a new class of miRNAs derived from the processing of nonprotein-coding regions of gene transcripts. The intronic miRNAs differ uniquely from previously described intergenic miRNAs in the requirement of RNA polymerase (Pol)-II and spliceosomal components for its biogenesis. Several kinds of intronic miRNAs have been identified in C. elegans, mouse, and human cells; however, their functions and applications have not been reported. Here, we show for the first time that intron-derived miRNA is not only able to induce RNAi in mammalian cells, but also in fish, chicken embryos, and adult mice cells, demonstrating the evolutionary preservation of this gene regulation system in vivo. These miRNA-mediated animal models provide artificial means to reproduce the mechanisms of miRNA-induced disease in vivo and will shed further light on miRNA-related therapies.

Key words

MicroRNA RNA interference RNA polymerase type II RNA splicing Intron RNA-induced gene-silencing complex Gene silencing in vivo 
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Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  1. 1.Division of Regenerative MedicineWJWU and LYNN Institute for Stem Cell ResearchSanta Fe SpringsUSA
  2. 2.Department of Cell and NeurobiologyUniversity of Southern CaliforniaLos AngelesUSA

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