VECTORS
Contributor Information
- Name Andrii Domanskyi, Piotr Chmielarz, Ilmari Parkkinen
- Institute University of Helsinki
- Primary citation N/A
Tool Details
- Tool name: Dicer activation reporter plasmid (DARA plasmid)
- Alternate names: pTO-miR19b-tdTom-19b_bind-WPRE-EGFP Dicer-activating reporter assay (DARA) plasmid
- Tool type: Vector
- Purpose: Plasmid for creation of DARA reporter cell line, by homologous recombination in HEK293 Flp-In T-REx cells
- Disease: Parkinson's disease; amyotrophic lateral sclerosis (ALS)
- Insert: Fluorescent proteins tdTomato and EGFP; human miR-19a-1 hairpin and binding site
- Application: High throughput screening of chemical compound libraries
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Cloning information: miRNA-19b-1 hairpin (184 bp) was amplified from human genomic DNA with the folowing primers and cloned into psiCHECK vector (digested with SalI/ApaI) using inFusion system.
miR19b_for: 5â gaggtagtgagtcgacGCCCAATCAAACTGTCCTGTTACTG
miR19b_rev: 5â catcagcgaaccgcgggcccGGTTTGAGTTTCCCTTACTTTTCTACAGAC -
Description: DARA plasmid was created for the Dicer activation reporter assay, which is used to screen compounds for miRNA biogenesis activation. Dicer has been implicated in certain cancers, ALS and Parkinsons Disease. The DARA plasmid was designed to express three main elements: 1) a primary fluorescent reporter protein, a tandem dimer Tomato (tdTomato) red fluorescent protein, which additionally has a nuclear localization sequence (NLS), 3â untranslated sequence (3âUTR) containing miRNA miR-19b binding site and a regulatory element, the Woodchuck virus post-transcriptional regulatory element (WPRE), to stabilize and increase expression, in its transcript; 2) a secondary fluorescent reporter protein, enhanced green fluorescent protein (EGFP) for normalization; and 3) a miR-19b hairpin designed to target 3âUTR of tdTomato.
When the miR-19b hairpin is expressed, it must be first processed by the Drosha/DGCR8 microprocessor complex, and then by Dicer to be loaded into RISC complex and inhibit tdTomato translation. When miRNA biogenesis is stimulated, the levels of mature miR-19b increase leading to stronger inhibition of tdTomato translation, and decrease of the ratio of fluorescence tdTomato/EGFP. - Research area: Cell biology
- Total vector size: 9990 bp
- Backbone size without insert: 4329 bp
- Insert size: 5661 bp
- Insert species: Aequorea victoria; Homo sapiens
- Promoter: Tet-ON CMV; SV40; TK-HSV
- Bacterial resistance: Ampicillin
- Selectable markers: Hygromycin
- For Research Use Only
Target Details
Application Details
- Application: High throughput screening of chemical compound libraries
Handling
- Storage buffer: Water
- Storage conditions: Store at -20°C
- Shipping conditions: +4°C or dry ice
- Plasmid amplification details: Stbl3 E.coli
- Plasmid copy number: High copy number
Documentation
Related Tools
References
- • N/A
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