KC-3431

293T-UAS-Luc2-Cell-Line

26445

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Background of 293T-UAS-Luc2-Cell-Line

The GAL4/upstream activating sequence (UAS) system is one of the most powerful tools for targeted gene expression. It is based on the properties of the yeast GAL4 transcription factor which activates transcription of its target genes by binding to UAS cis-regulatory sites. In Drosophila, the two components are carried in separate lines allowing for numerous combinatorial possibilities. The driver lines provide tissue-specific GAL4 expression and the responder lines carry the coding sequence for the gene of interest under the control of UAS sites.

Specifications

Catalog Number	KC-3431
Cell Line Name	293T-UAS-Luc2-Cell-Line
Host Cell Line	293T
Description	Stable HEK293T cell line expressing exogenous luciferase under the control of UAS response element
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	DMEM + 20% FBS + 10%DMSO
Propagation Medium	DMEM + 10%FBS + 150μg/mL Hygromycin B
Selection Marker	Hygromycin B
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

293T-UAS-Luc2 cell line was generated using a lentiviral vector expressing luciferase under the control of UAS response element.

Characterization

Figure 1. DNA vector expressing GAL4-VP16 was transfected into 293T-UAS-Luc2 cells by transient transfection method, then readout with Bright-lite™ Luciferase Assay system after 24 hours.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS, 150μg/ml Hygromycin B)in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage

References

Busson D, Pret AM. GAL4/UAS targeted gene expression for studying Drosophila Hedgehog signaling. Methods Mol Biol. 2007;397:161-201. doi: 10.1007/978-1-59745-516-9_13. PMID: 18025721.