The shMIMIC Inducible microRNA vectors incorporate the Tet-On® 3G bipartite induction system a 3rd-generation Tet-inducible system significantly improved and optimized for minimal basal expression (lowest leakiness) and potent activation upon induction (Zhou X, et al., Gene Therapy 13, 1382 (2006) and Loew R, et al., BMC Biotechnol. 10, 81 (2010)). The Tet-On® 3G Inducible System permits tightly controlled shMIMIC microRNA expression and study of gene function in vivo and in vitro with unprecedented precision.
Assay Development and Optimization: Establish optimal experimental conditions, including those for a) lentiviral transduction and b) screening parameters, such as selective pressure and time between collection of reference and experimental samples.
Primary Screen: A stable population of cells expressing single integrands of constructs are created by transducing lentiviral pools at low MOIs. Transduced cells are then split into reference and experimental populations for application of a selective pressure that induces the phenotype of interest. Genomic DNA (gDNA) is then isolated from reference and experimental populations of transduced cells. Illumina-adapted primers and Phusion Hot-Start II High Fidelity DNA Polymerase are used to PCR amplify integrated construct sequences and add Illumina flow-cell binding sequences. The resulting amplicons are run on Illumina platform sequencers, using the sequencing primers provided.
Hit Identification and Follow-up: Construct sequences are identified in reference and experimental libraries. Constructs that are enriched or depleted during the screen are identified as hits, and the genes that they target are identified. Hits can be confirmed and studied further using individual constructs that can be ordered from the Dharmacon catalog collection.
SMARTchoice Inducible Non-targeting Controls were used to transduce the indicated cell types at MOI = 0.3. 24 h after transduction, expression of the non-targeting shRNA and PuroR was induced with 1 µg/mL doxycycline. After 48 h of culture in the presence of doxycycline, cells were stained with Hoescht-33342 and nuclei (blue) and TurboGFP (green) were imaged. While ~30% of the cells in the field have been transduced, some images may appear to contain fewer than 30% TurboGFP-positive cells due to low TurboGFP expression, indicative of low constitutive promoter activity, in a particular cell type.
HUVEC (primary human umbilical vein endothelial) cells were transduced at MOI = 0.2 with the indicated SMARTvector Inducible Lentiviral shRNA vectors carrying a non-targeting control shRNA (NTC) or shRNAs directed against RHOA or GAPDH. Cells were selected with 1.5 µg/mL puromycin for 72 hours. shRNA expression was then induced with 0, 0.01, 0.1, 1.0 and 10 µg/mL doxycycline, respectively, and mRNA was harvested 72 hours after dox-induction. Target gene silencing was measured by RT-qPCR relative to the PPIB reference gene. Gene silencing is expressed relative to the vector- and doxycycline dosage-matched NTC cell population.
The Human-Mouse Conserved microRNA pooled library is useful for researchers that need ultra-high fold coverage for subtle phenotypes, or who may not have enough cells (e.g. primary cells) for a larger screen and wish to focus on highly conserved microRNAs. It can be used in either human or mouse models, and translates well to in vivo applications.
It includes 386 total mature microRNAs:
302 human and mouse matures that are identical
35 mouse matures in broadly conserved families that are not identical to human
49 human matures in broadly conserved families that are not identical to mouse
Broadly conserved families as defined at www.targetscan.org