Dicer activation reporter cell line

Tool Details

*FOR RESEARCH USE ONLY (for other uses, please contact the licensing team)

• Name: Dicer activation reporter cell line
• Alternate name: DARA cells, HEK293-Dicer2.0-miR19b Dicer-activating reporter assay (DARA) cell line
• Research fields: Cancer;Cell biology
• Tool sub type: Continuous
• Parental cell: FLP-IN HEK293 T-Rex cells (ThermoFisher Scientific, Waltham, MA)
• Organism: Human
• Gender: Female
• Tissue: Kidney
• Donor: Original HEK 293 cells were obtained from a single, aborted or miscarried fetus, the precise origin of which is unclear
• Disease: Parkinson's disease; ALS
• Morphology: Epithelial
• Growth properties: Adherent
• Model: Knock-In
• Model description: Stable transfection of FLP-IN HEK293 T-REX cells. After T-Rex cells (ThermoFisher Scientific, Waltham, MA) reached 80-90% confluency, they were re-plated into Greiner CellStar dishes in DMEM supplemented with 10% FBS and 100 ?g/ml Normocin. 1 ?g of pTO-miR19b-tdTom-19b_bind-WPRE-EGFP was transfected simultaneously with 5 ?g of flippase (FLP) expressing plasmid pOG44 using 1 ?g/?l polyethylenimine (PEI) (4:1 ratio of PEI/DNA) as the transfection reagent, diluted in OptiMEM (ThermoFisher Scient...
• Crispr: No
• Description: The cell line was created for Dicer activation reporter assay that will be used to screen small molecules and other compounds for miRNA biogenesis activation. The reporter construct to measure Dicer activity is an expression vector 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), 3’ untranslated sequence (3’UTR) containing miRNA miR-19b binding site and a regulatory element, the Woodchuck virus posttranscriptional 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 3’UTR of tdTomato. The addition of NLS to tdTomato is an improvement allowing us to not only spectrally but also spatially separate reporter (tdTomato) from normalizer (EGFP) fluorescence. Furthermore such separation allows for inclusion of additional reporters in the future.
• Application: High throughput screening of chemical compound libraries
• Production details: The cells were plated into Greiner CellStar 96-well clear-bottom plates for a density of ca 10000 cells/well in DMEM supplemented with 10% FBS and 100 ?g/ml Normocin and incubated for 24 hours in a humidified incubator (37C, 5% CO2).
• Biosafety level: 1

Applications

• Application: High throughput screening of chemical compound libraries
• Application notes: This cell-based Dicer activity reporter assay can be used for high throughput screening of chemical compound libraries. Also, by changing the sequence of miRNA binding site and miRNA hairpin, the assay can be applied to study the effects of Dicer stimulation on the biogenesis of particular miRNAs. Those compounds can be patented for a potential treatment of PD, but also other neurodegeneration diseases such as ALS and Alzheimer’s disease that may share similar disease mechanisms affecting miR...

Handling

• Growth medium: DMEM (pH 7.4) supplemented with 10% fetal bovine serum (FBS), 100 ?g/ml Normocin (InvivoGen, San Diego, USA), 15 ?g/ml Blasticidin HCl and 100 ?g/ml Hygromycin B.
• Temperature: 37° C
• Atmosphere: 5% CO2
• Shipping conditions: Dry ice
• Storage medium: DMEM (pH 7.4) supplemented with 10% fetal bovine serum (FBS), 100 ?g/ml Normocin (InvivoGen, San Diego, USA), 15 ?g/ml Blasticidin HCl and 100 ?g/ml Hygromycin B + 5% sterile DMSO
• Storage conditions: Liquid Nitrogen, approx. 5x10^6 cells in a vial
• Initial handling information: 1) Add 10 mL pre-warmed cell culture media to 15 mL Falcon tube; 2) Rapidly thaw frozen vial in water bath at 37°C for 30-60 sec; 3) Pour cells from the vial to 15 mL Falcon tube (from Step 1); 4) Spin down at 130x g at room temperature for 5 min; 5) Carefully aspirate media; 6) Carefully resuspend cell pellet in 10 ml fresh pre-warmed media; 7) Plate on Petri dish or cell culture flask; 8) Incubate at 37C in 5% CO2 incubator
• Subculture routine: Replated at 1:5 dilution every 3-4 days
• Cultured in antibiotics: Normocin, Blasticidin HCl and Hygromycin
• Mycoplasma free: Yes

Related Tools

• Related tools: Dicer activation reporter plasmid (DARA plasmid) (161705)