|
| Status |
Public on May 07, 2015 |
| Title |
Engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus [miRNA] |
| Organism |
Homo sapiens |
| Experiment type |
Non-coding RNA profiling by array
|
| Summary |
Short hairpin RNA (shRNA) expression strategies that allow safe and persistent target mRNA knockdown are key to the success of many in vitro or in vivo RNAi applications. Here, we propose a novel solution which is expression of a promoterless miRNA-adapted shRNA (shmiRNA) from an engineered genomic miRNA locus. For proof-of-concept, we genetically “vaccinated” liver cells against a human pathogen, by using TALEns or CRISPR to integrate an anti-hepatitis C virus (HCV) shmiRNA into the liver-specific miR-122/hcr gene. Reporter assays and qRT-PCR confirmed anti-HCV shmiRNA expression as well as miR-122 integrity and functionality. Specificity and safety of shmiRNA integration were validated via PCR, cDNA and miRNA profiling, and whole genome sequencing. A subgenomic HCV replicon and a full-length reporter virus, but not a Dengue virus control, were significantly impaired in the modified cells. Our original combination of DNA engineering and RNA expression technologies should benefit numerous applications, from basic miRNA research, to human cell and gene therapy
|
| |
|
| Overall design |
Four Huh7 cells lines at 3 different passages were analyzed. The reference cell line was Huh7 wild type cells (WT). The other three cell lines had an integration of an anti-HCV shmiRNA in the hcr locus and miR-122 intact (T2 31.3) or mutated (TS 30.20 and U6 20.16). RNA was extracted from three different passages.
|
| |
|
| Contributor(s) |
Senis E, Grimm D |
| Citation(s) |
27614072 |
| Submission date |
May 06, 2015 |
| Last update date |
Dec 13, 2017 |
| Contact name |
Tobias Bauer |
| Organization name |
German Cancer Research Center
|
| Department |
Theoretical Bioinformatics
|
| Lab |
eilslabs
|
| Street address |
Im Neuenheimer Feld 280
|
| City |
Heidelberg |
| ZIP/Postal code |
69120 |
| Country |
Germany |
| |
|
| Platforms (1) |
| GPL18044 |
Agilent-046064 Unrestricted_Human_miRNA_V19.0_Microarray (Feature Number version) |
|
| Samples (12)
|
|
| This SubSeries is part of SuperSeries: |
| GSE68638 |
Engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus |
|
| Relations |
| BioProject |
PRJNA283227 |
Less...
More...