Multi-miRNA hairpin method that improves gene knockdown efficiency and provides linked multi-gene knockdown

Abstract

A number of natural microRNA (miRNA) hairpins have been found in clusters of multiple identical or different copies, suggesting that effects of miRNAs can be enhanced and multiple genes can be regulated together by encoding multiple miRNA hairpins in a single transcript. Here, we report a simple and effective artificial multi-hairpin method that stimulates production of mature 22-nucleotide small RNAs from modified miRNA hairpins, improves gene knockdown over single-hairpin constructs, and provides linked multi-gene knockdowns.

The Acetylase/Deacetylase Couple CREB-binding Protein/Sirtuin 1 Controls Hypoxia-inducible Factor 2 Signaling

“…For CBP knockdown/rescue, we injected 3 ϫ 10 11 particles of PPN HIF-2␣:HA or control (empty) adenovirus that also express control or mouse CBP shRNA (PPN HIF-2␣:HA/shRNA adenovirus), plus 3 ϫ 10 11 particles of either control adenovirus or adenovirus expressing Myc-tagged shRNA-resistant (resist) wild-type (WT) or acetyltransferase activity mutant (HAT) mouse CBP. The PPN HIF-2␣:HA/shRNA adenovirus expresses PPN HIF-2␣:HA followed by an internal ribosome entry site with a downstream DsRed cassette containing multiple shRNA at the 3Ј end of the DsRed cDNA (17). The resist CBP cDNA maintains amino acid identity but is designed to disrupt binding of the shRNA that targets endogenous mouse CBP.…”

Site-directed, virus-free, and inducible RNAi in embryonic stem cells

“…For multiple shRNA-mir hairpin strategy, the minimal miR sequence that was required for shRNA processing was amplified (Ϸ150 bp) for individual shRNA-mir hairpin to minimize the cloning size. Primers were designed as previously described (23), except that XhoI, KpnI, SphI, and XbaI restriction sites were incorporated and used for cloning the multi-shRNA-mir cassette into the pBluescript vector. The final XhoI-XbaI fragments were released from the pBluescript vector and cloned into a pLox vector previously digested with XhoI and XbaI.…”

“…The incorporation of multiple miRNA hairpins in a single vector reportedly improves the efficiency of target gene knockdown (23). To test whether knockdown efficiency in our system could be enhanced, Ainv15 ES cells were transfected with shRNA-mir vectors containing a single (siNanog), double (diNanog), or multiple (miNanog) shRNAs directed against Nanog.…”

Diagnostic and Biological Significance of KIR Expression Profile Determined by RNA-Seq in Natural Killer/T-Cell Lymphoma

“…We then generated a more effective retroviral shRNA construct (Supplemental Figure S2, C and D) by tandemly linking two KIR2DL4 shRNAemiRNAs as described previously. 23 We observed a more robust decrease in the percentage of GFP þ cells in KIR2DL4 doubleshRNAetransduced NK92 ( Figure 2B) or KHYG1 ( Figure 2C) cells compared to single-shRNAetransduced cells. Next, we evaluated apoptosis by quantifying the percentage of annexin V-phycoerythrin þ /GFP þ KHYG1 cells transduced with the empty vector or KIR2DL4 doubleshRNA, and observed moderately higher annexin V positivity in cells with KIR2DL4 knockdown, suggesting that an increased rate of apoptosis may be involved in the negative selection ( Figure 2D).…”