Optimized miR-30 shRNAmir backbone (miR-E)
Optimized variant of the miR-30 shRNAmir backbone that restores a conserved ACNNC motif 3′ of the basal stem by moving the EcoRI cloning site into a nonconserved region and adjusting the opposing 5′ scaffold. It increases pri-miRNA processing, raising mature shRNA levels 7- to 32-fold and single-copy knockdown potency relative to miR-30.
Origin: miR-30 backbone with EcoRI site relocated
Characteristics
The core stem-loop is unchanged, so existing miR-30 shRNAs convert to miR-E by PCR subcloning with XhoI/EcoRI. Point mutations at three conserved positions of the -ACNNC- motif reduce knockdown to or below miR-30 levels. At single copy in NIH 3T3 cells, miR-E lowers pri-miRNA levels and raises mature small RNA 7- to 32-fold, about tenfold for Ren.713 and Pten.1523, while mature shRNA stays below the most abundant endogenous miRNAs and endogenous miRNA levels are unchanged (r = 0.99).
Applications: Single-copy shRNA expression in mouse fibroblast, hepatocyte, endothelial and kidney lines (NIH 3T3, BNL CL.2, SVEC4-10, TCMK-1) and chicken embryonic fibroblasts. Seven MOLM-13 essential-gene shRNAs depleted cells faster in miR-E than miR-30, and all 11 Dnmt3a shRNAs exceeded 80% knockdown. Built into constitutive and T3G-driven Tet-regulated retroviral (pMSCV, pQCXIX) and lentiviral (pRRL) vectors.
Limitations: Higher pri-miRNA processing lowers expression of a linked GFP reporter and reduces retroviral titers, so miR-E vectors package less efficiently than miR-30 vectors. Adding the endogenous loop, R1B1 or R2B2 features gives no further gain. Mirimus, which employs the first author, has applied for intellectual property protection of some of the described methods.
References
- Fellmann et al. (2013). An optimized microRNA backbone for effective single-copy RNAi. Cell Rep - Fellmann 2013 miR-E Backbone