Abstract
The relative ease of identifying microRNAs and their increasing recognition as important regulators of organogenesis motivate the development of methods to efficiently assess microRNA function during organ morphogenesis. In this context, embryonic organ explants provide a reliable and reproducible system that recapitulates some of the important early morphogenetic processes during organ development. Here we present a method to target microRNA function in explanted mouse embryonic organs. Our method combines the use of peptide-based nanoparticles to transfect specific microRNA inhibitors or activators into embryonic organ explants, with a microRNA pulldown assay that allows direct identification of microRNA targets. This method provides effective assessment of microRNA function during organ morphogenesis, allows prioritization of multiple microRNAs in parallel for subsequent genetic approaches, and can be applied to a variety of embryonic organs.
Highlights
The relative ease of identifying microRNAs and their increasing recognition as important regulators of organogenesis motivate the development of methods to efficiently assess microRNA function during organ morphogenesis
Methods for in vivo miRNA functional perturbation include the injection of chemically modified miRNA inhibitors[2], referred to here as antagomirs, and viral infections of DNA constructs expressing miRNA inhibitors[3], both of which are limited to postnatal developmental stages and can generate systemic off-target effects
We found that Nanoparticle-Forming Solution (NFS) was comparatively more efficient than Liposome-Forming Solution (LFS) in transfecting Antagomir-Cy3 into explanted E13.5 submandibular salivary glands (SMGs) and E11.5 lungs cultured on Nuclepore floating filters
Summary
The relative ease of identifying microRNAs and their increasing recognition as important regulators of organogenesis motivate the development of methods to efficiently assess microRNA function during organ morphogenesis. Our method combines the use of peptide-based nanoparticles to transfect specific microRNA inhibitors or activators into embryonic organ explants, with a microRNA pulldown assay that allows direct identification of microRNA targets This method provides effective assessment of microRNA function during organ morphogenesis, allows prioritization of multiple microRNAs in parallel for subsequent genetic approaches, and can be applied to a variety of embryonic organs. Antagomirs can incorporate a chemical modification termed Locked Nucleic Acid (LNA)[2,15], which consists of a 2′ , 4′ methylene-bridge in the ribose that forms a bicyclic nucleotide with higher affinity binding to the complementary miRNA target This allows the use of short LNA-modified oligonucleotides in multiple applications including miRNA in situ hybridization[16] and knockdown studies[15].
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