Hybrid Cytomegalovirus-U6 Promoter-based Plasmid Vectors Improve Efficiency of RNA Interference in Zebrafish

Abstract

Short hairpin RNA (shRNA) directed by RNA polymerase III (Pol III) or Pol II promoter was shown to be capable of silencing gene expression, which should permit analyses of gene functions or as a potential therapeutic tool. However, the inhibitory effect of shRNA remains problematic in fish. We demonstrated that silencing efficiency by shRNA produced from the hybrid construct composed of the CMV enhancer or entire CMV promoter placed immediately upstream of a U6 promoter. When tested the exogenous gene, silencing of an enhanced green fluorescent protein (EGFP) target gene was 89.18±5.06% for CMVE-U6 promoter group and 88.26 ± 6.46% for CMV-U6 promoter group. To test the hybrid promoters driving shRNA efficiency against an endogenous gene, we used shRNA against no tail (NTL) gene. When vectorized in the zebrafish, the hybrid constructs strongly repressed NTL gene expression. The NTL phenotype occupied 52.09± 3.06% and 51.56±3.68% for CMVE-U6 promoter and CMV-U6 promoter groups, respectively. The NTL gene expression reduced 82.17±2.96% for CMVE-U6 promoter group and 83.06±2.38% for CMV-U6 promoter group. We concluded that the CMV enhancer or entire CMV promoter locating upstream of the U6-promoter could significantly improve inhibitory effect induced by the shRNA for both exogenous and endogenous genes compared with the CMV promoter or U6 promoter alone. In contrast, the two hybrid promoter constructs had similar effects on driving shRNA.

Pseudotyped Adeno-Associated Virus 2/9-DeliveredCCL11shRNA Alleviates Lung Inflammation in an Allergen-Sensitized Mouse Model

“…Although shRNA expression is usually directed by polymerase III promoters, such as U6 or H1, better gene inhibitory efficacy has been demonstrated with the hybrid CMV/ U6 or CMV/H1 promoters, compared to single U6 or H1 promoter (Hassani et al, 2007;Su et al, 2008). Similar results were also observed in the current study.…”

“…Small hairpin RNA (shRNA) can be efficiently transcribed into small interfering RNA in mammalian cells via the U6 or hybrid CMV/U6 promoter. The hybrid promoters, CMV/U6 or CMV/H1, showed better shRNA expression efficiency than U6 or H1 promoter (Hassani et al, 2007;Su et al, 2008). Adeno-associated virus (AAV), a single-stranded DNA virus that belongs to the Parvoviridae family, is characterized by its nonpathogenic nature, nonintegrating episomal vector genome, and ability to confer stable gene expression (Daya and Berns, 2008).…”

Vascular Smooth Muscle-Specific Knockdown of the Noncardiac Form of the L-Type Calcium Channel by MicroRNA-Based Short Hairpin RNA as a Potential Antihypertensive Therapy

“…In vivo vascular selectivity, time course of gene transduction, efficiency of siRNA production, decrease in Ca V 1.2 expression and function in small resistance arteries, and corresponding changes in blood pressure all need to be carefully monitored and analyzed. A recent study showing in vivo application of shRNA expression cassette in zebrafish is encouraging (Su et al, 2008), and we are currently working on the in vivo application of our constructs.…”

Oligonucleotide-Based Tools for Studying Zebrafish Development

“…Constitutive shRNA expression through the use of U635 or CMV36 promoters induced a modest knockdown of GFP, Ntla, and VEGF-A protein levels at plasmid doses that did not cause toxic off-target effects 37. Driving shRNA expression through a hybrid U6/CMV promoter can enhance RNA interference efficiency, reducing Ntla protein to approximately 20% of wildtype levels and producing a ntla mutant phenotype in about half of the injected embryos 38. Transgenic zebrafish that stably express shRNAs through a pri-miR-30-derived stem-loop precursor have also been generated 39.…”