title: Creating conditional dual fluorescence labeled transgenic animals for studying function of small noncoding RNAs.
abstract: Because the function of most noncoding (nc) RNAs is unknown, Cre-lox transgenic mice are useful tools to determine their functions in a tissue or developmental stage-specific manner. However, the technology faces challenges because expression of ncRNA-transgene lacks protein product. No antibody or peptide-tag can be used to trace ncRNA expression in mouse tissues in real time. Furthermore, transgene integration at different locus or orientations in the genome may result in recombination of genomic fragments in the Cre-lox system. Establishing a reliable method that can be used to determine the precise copy number and orientation of the transgene is critical to the field. We developed a fast and straightforward method to determine ncRNA-transgene copy number, orientation, and insertion site in the genome. Furthermore, upon tissue-specific expression of ncRNA, a Cre-loxP-mediated dual-fluorescence expression system facilitates fluorescence signal switching from green to red, which enables real-time monitoring of ncRNA expression by fluorescence signals. As proof of concept, we demonstrate that after microRNA (miRNA)-Flox mice crossed with Col2a1-Cre mice, miRNA transgene expression could be detected successfully by red fluorescence signals in various cartilaginous tissues. This method of creating small ncRNA transgenic mice facilitates both tissue-specific ncRNA expression and real-time visualization of its expression. It is particularly suitable for in vivo studies of the functional roles and lineage tracing of small ncRNA.
authors.affiliation: a Department of Orthopedics , Warren Alpert Medical School of Brown University and Rhode Island Hospital , Providence , RI , USA.
authors.forename: Kun
authors.initials: K
authors.lastname: Yang
authors.affiliation: a Department of Orthopedics , Warren Alpert Medical School of Brown University and Rhode Island Hospital , Providence , RI , USA.
authors.forename: Yun
authors.initials: Y
authors.lastname: Gao
authors.affiliation: a Department of Orthopedics , Warren Alpert Medical School of Brown University and Rhode Island Hospital , Providence , RI , USA.
authors.forename: Mingfu
authors.initials: M
authors.lastname: Yang
authors.affiliation: b Department of Biochemistry and Molecular Pharmacology , University of Massachusetts Medical School , Worcester , MA , USA.
authors.forename: Zuoshang
authors.initials: Z
authors.lastname: Xu
authors.affiliation: a Department of Orthopedics , Warren Alpert Medical School of Brown University and Rhode Island Hospital , Providence , RI , USA.
authors.forename: Qian
authors.initials: Q
authors.lastname: Chen
mesh_terms: D000818:Animals; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005786:Gene Expression Regulation; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence / Q000379:methods; D058727:RNA, Small Untranslated / Q000096:biosynthesis / Q000235:genetics; D019076:Transgenes
chemical_list: D058727:RNA, Small Untranslated
keywords: Droplet digital PCR; dual fluorescence switching system; microRNA; noncoding RNA; transgene copy number; transgenic mice