Characterization and Comparison of Chicken U6 Promoters for the Expression of Short Hairpin RNAs

Abstract

RNA interference (RNAi) is a powerful method of sequence-specific gene knockdown that can be mediated by DNA-based expression of short hairpin RNA (shRNA) molecules. A number of vectors for expression of shRNA have been developed with promoters for a small group of RNA polymerase III (pol III) transcripts of either mouse or human origin. To advance the use of RNAi as a tool for functional genomic research and future development of specific therapeutics in the chicken species, we have developed shRNA expression vectors featuring chicken U6 small nuclear RNA (snRNA) promoters. These sequences were identified based on the presence of promoter element sequence motifs upstream of matching snRNA sequences that are characteristic of these types of pol III promoters. To develop suitable shRNA expression vectors specifically for chicken functional genomic RNAi applications, we compared the efficiency of each of these promoters to express shRNA molecules. Promoter activity was measured in the context of RNAi by targeting and silencing the reporter gene encoding the enhanced green fluorescent protein (EGFP). Plasmids containing one of four identified chicken U6 promoters gave a similar degree of knockdown in DF-1 cells (chicken); although, there was some variability in Vero cells (monkey). Because the chicken promoters were not stronger than the benchmark mouse U6 promoter, we suggest that the promoter sequence and structure is more important in determining efficiency in vitro rather than its species origin.

βig-h3 Potentiates the Profibrogenic Effect of TGFβ Signaling on Connective Tissue Progenitor Cells Through the Negative Regulation of Master Chondrogenic Genes

“…For big-h3 loss-of-function experiments, limb mesodermal cells were transfected with a short-hairpin RNAi for big-h3 (shbig-h3) cloned into the pcU6-1-shRNA (a generous gift of Dr. Tim J. Doran) as described by Wise et al 25 The limb mesodermal cells were electroporated as mentioned above. The level of gene silencing was evaluated by Q-PCR and by ELISA.…”

Short Hairpin RNA-Mediated Gene Silencing

“…These nine loci are dispersed throughout the genome and fi ve were found to have associated promoter regions, each with different transcriptional activities. These fi ndings spurred the characterization and comparison of U6 promoters for shRNA transcription from a variety of other species including bovine ( 69,70 ) , chicken ( 71,72 ) , fugu ( 73 ) , zebra fi sh ( 74 ) , sheep ( 75 ) , and pig ( 76 ) .…”

Systemic Remodeling of the Redox Regulatory Network due to RNAi Perturbations of Glutaredoxin, Thioredoxin, and Glucose-6-phosphate Dehydrogenase

“…All of the shRNA cell lines used for this study had undergone at least three passages from the lentiviral infection and puromycin selection before characterization. Because of the advantages to using shRNA, this method of examining redox regulation of cellular processes has been increasingly used to study effects of NAD(P)H supply, glutaredoxin, thioredoxin, and associated reductases [16][17][18][19][20][21][22]. Many of these studies rely on perturbation of the target protein in order to observe a phenotypic change, such as sensitivity to diamideinduced oxidative stress [19] or increased cellular senescence [22], and rely on the assumption that the rest of the antioxidant enzyme system remains intact.…”

“…Non-overlapping sequences of short hairpin RNA (shRNA) which have variable efficiency of interference can be exploited to generate "epi-allelic" cell lines with a range of protein silencing [13,14]. As RNAi is utilized with greater frequency to investigate the role of oxidative protein thiol modifications in cellular function [15][16][17][18][19][20][21][22], it is important to consider the specificity of RNAi perturbations with respect to intracellular oxidant sources and sinks. The introduction of viral particles induces oxidative stress and can alter cellular antioxidant levels; this alteration of cellular protein levels [23,24] may have unexpected redox-related consequences beyond potential off-target silencing, consequences that may substantially alter the redox capacity of the knockdown cells.…”