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.
Highlights
MicroRNAs are 19- to 24-nt small non-coding RNAs that are processed from primary miRNA through intermediate stem-loop precursor miRNA structures[1,2,3]
Placental trophoblasts, which are bathed in maternal blood in human and murine pregnancy, support pregnancy by regulating the exchange of gas, nutrients, and waste between the mother and foetus, producing hormones that are essential for pregnancy and providing immunological defence to the allogeneic foetus
The control blastocysts were incubated in a CO2 incubator for 3 days after exposure to each lentiviral construct and enhanced green fluorescent protein (EGFP) signal was observed exclusively in the trophectoderm of all blastocysts between 24 and 72 h (Fig. 1a, left)
Summary
MicroRNAs (miRNAs) are 19- to 24-nt small non-coding RNAs that are processed from primary miRNA (pri-miRNA) through intermediate stem-loop precursor miRNA (pre-miRNA) structures[1,2,3]. Several groups have recently introduced an efficient method for murine placenta-specific gene expression[24,25,26], in which lentivirus vectors are essential due to their potent transduction efficiency in the blastocyst’s trophectoderm layer. This system has been recently deployed to address trophoblast gene function and placental pathophysiology[27,28,29,30]. Building on recent advances in the field, as well as our findings described here, we designed a novel lentivirus-based miRNA expression system for in vivo use We tested this system by transfecting mouse placentas with diverse types of miRNAs. As expected, we found that inverted intronic miRNAs are expressed at a much higher level than forward inserts. We showed that members of the poly(A)-binding protein (PABP) family are germane for lentivirus-expressed miRNA prior to genomic integration
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