Harnessing Intronic microRNA Structures to Improve Tolerance and Expression of shRNAs in Animal Cells

Methods and Protocols

Timothy J. Doran¹  Mark L. Tizard¹  Arjun Challagulla¹  Kristie A. Jenkins¹  David M. Cahill² 

[1]CSIRO Health and Biosecurity, Australian Centre for Disease Preparedness, Geelong, VIC 3219, Australia

[2]School of Life and Environmental Sciences, Deakin University, Geelong, VIC 3217, Australia

关键词: avian;    RNAi;    promoters;    transgenic;    miRNAs;    shRNA;   

DOI  :  10.3390/mps5010018

来源: DOAJ

【 摘 要 】

Exogenous RNA polymerase III (pol III) promoters are commonly used to express short hairpin RNA (shRNA). Previous studies have indicated that expression of shRNAs using standard pol III promoters can cause toxicity in vivo due to saturation of the native miRNA pathway. A potential way of mitigating shRNA-associated toxicity is by utilising native miRNA processing enzymes to attain tolerable shRNA expression levels. Here, we examined parallel processing of exogenous shRNAs by harnessing the natural miRNA processing enzymes and positioning a shRNA adjacent to microRNA107 (miR107), located in the intron 5 of the Pantothenate Kinase 1 (PANK1) gene. We developed a vector encoding the PANK1 intron containing miR107 and examined the expression of a single shRNA or multiple shRNAs. Using qRT-PCR analysis and luciferase assay-based knockdown assay, we confirmed that miR30-structured shRNAs have resulted in the highest expression and subsequent transcript knockdown. Next, we injected Hamburger and Hamilton stage 14–15 chicken embryos with a vector encoding multiple shRNAs and confirmed that the parallel processing was not toxic. Taken together, this data provides a novel strategy to harness the native miRNA processing pathways for shRNA expression. This enables new opportunities for RNAi based applications in animal species such as chickens.

【 授权许可】

Unknown