Gam JJ, Babb J, Weiss R
Nat Commun. 2018 Jun 22;9(1):2430. doi: 10.1038/s41467-018-04575-0. PubMed Article
This collection of plasmids consists of miRNA sensors used to measure miRNA activity in mammalian cells. The plasmids encode constitutive expression of two fluorescent proteins from the hEF1a promoter, one of which (mKate2) is regulated by miRNA target sites in the 3’ UTR while the other (EBFP2) serves as a transfection marker. Target sites consist of four repeats of the sequence perfectly complementary to the mature human miRNA sequence. When transiently transfected or genomically integrated into cells, miRNAs with detectable activity will downregulate mKate2 fluorescence relative to EBFP2 fluorescence, measurable using flow cytometry or fluorescence microscopy.
Additionally, this plasmid collection can serve as a low-cost source for miRNA target sites. Target sites can be extracted via PCR to generate sequences containing distinct overhangs for restriction ligation or Golden Gate assembly into other miRNA sensor circuits. Several backbone plasmids are provided for researchers to assemble new miRNA sensors for novel or candidate miRNAs, or those for other species.Weiss Lab miRNA Low Sensor Library Protocols (12.6 KB)