title: Determinants of effective lentivirus-driven microRNA expression in vivo.
abstract: Manipulation of microRNA (miRNA) levels, including overexpression of mature species, has become an important biological tool, even motivating miRNA-based therapeutics. To assess key determinants of miRNA overexpression in a mammalian system in vivo, we sought to bypass the laborious generation of a transgenic animal by exploiting placental trophoblast-specific gene manipulation using lentiviral vectors, which has been instrumental in elucidating trophoblast biology. We examined the impact of several key components of miRNA stem loops and their flanking sequences on the efficiency of mature miRNA expression in vivo. By combining established and novel approaches for miRNA expression, we engineered lentivirus-driven miRNA expression plasmids, which we tested in the mouse placenta. We found that reverse sense inserts minimized single-strand splicing and degradation, and that maintaining longer, poly-A-containing arms flanking the miRNA stem-loop markedly enhanced transgenic miRNA expression. Additionally, we accomplished overexpression of diverse mammalian, drosophila, or C. elegans miRNAs, either based on native context or using a "cassette" replacement of the mature miRNA sequence. Together, we have identified primary miRNA sequences that are paramount for effective expression of mature miRNAs, and validated their role in mice. Principles established by our findings may guide the design of efficient miRNA vectors for in vivo use.
authors.affiliation: Magee-Womens Research Institute, Department of Obstetrics, Gynaecology and Reproductive Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, 15213 USA.
authors.forename: Takuya
authors.initials: T
authors.lastname: Mishima
authors.affiliation: Magee-Womens Research Institute, Department of Obstetrics, Gynaecology and Reproductive Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, 15213 USA.
authors.forename: Elena
authors.initials: E
authors.lastname: Sadovsky
authors.affiliation: Magee-Womens Research Institute, Department of Obstetrics, Gynaecology and Reproductive Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, 15213 USA.
authors.forename: Margaret E
authors.initials: ME
authors.lastname: Gegick
authors.affiliation: Magee-Womens Research Institute, Department of Obstetrics, Gynaecology and Reproductive Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, 15213 USA.
authors.forename: Yoel
authors.initials: Y
authors.lastname: Sadovsky
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017173:Caenorhabditis elegans / Q000235:genetics; D019070:Cell Lineage; D004331:Drosophila melanogaster / Q000235:genetics; D005260:Female; D005333:Fetus / Q000378:metabolism; D015870:Gene Expression; D007438:Introns / Q000235:genetics; D016086:Lentivirus / Q000378:metabolism*; D051379:Mice; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D010920:Placenta / Q000378:metabolism; D039122:Poly(A)-Binding Protein I / Q000378:metabolism; D011247:Pregnancy; D012326:RNA Splicing / Q000235:genetics; D014161:Transduction, Genetic; D014327:Trophoblasts / Q000166:cytology
chemical_list: D035683:MicroRNAs; D039122:Poly(A)-Binding Protein I