Characteristics of inducible miR-122 expression with Rheoswitch. a. RSL1 structure and schematic representation of the Rheoswitch system. b-f: Northern blot analyses of total RNA from non-induced (DMSO) and induced (RSL1) cells treated as indicated for each panel. b. HEK293-A7 cells transiently transfected with pNEBRX-1 containing a genomic miR-122 fragment. c. Top panel: Production of miR-122 guide vs. passenger strand: stably transformed NIH3T3-47-miR122 cells were treated as indicated. Bottom panel: RSL1 concentration-dependent expression of miR-122. Cells were treated with DMSO (lane 1) or increasing concentrations of RSL1 (50 nM, 500 nM or 5 μM, lanes 2-4)Probe: miR-122 guide strand. d. Time course of induction: RNA was prepared from cells at times indicated, beginning 2 hours after addition of DMSO (left) or RSL1 (right). Note that although the 24 h non-induced cell RNA sample is overloaded no miR-122 guide strand is detected. e. miR-122 expression in human hepatocarcinoma derived HepG2 and Huh7 cell lines. f. Switch on/off properties. Top: schematic representation of treatment regimen and sampling times. Each row (I to IV) of the scheme corresponds to a row on the northern blot below. Time is indicated as days elapsed, + RSL1 (shaded) and - RSL1 (clear). Bottom: Northern blot of total RNA hybridized with miR-122 guide strand probe (left) or U6 control (right). See Methods for probe sequences.
Shea and Tzertzinis BMC Biotechnology 2010 10:76 doi:10.1186/1472-6750-10-76