KC-3471

293T-NFAT-Luc2-GNRHR Cell Line

34412

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Background of 293T-NFAT-Luc2-GNRHR Cell Line

GNRHR (Gonadotropin Releasing Hormone Receptor) is a Protein Coding gene. Diseases associated with GNRHR include Hypogonadotropic Hypogonadism 7 With Or Without Anosmia and Hypogonadotropic Hypogonadism. Among its related pathways are GPCR downstream signalling and Class A/1 (Rhodopsin-like receptors). Gene Ontology (GO) annotations related to this gene include G protein-coupled receptor activity and gonadotropin-releasing hormone receptor activity.

Specifications

Catalog Number	KC-3471
Cell Line Name	293T-NFAT-Luc2-GNRHR Cell Line
Host Cell Line	293T-NFAT-Luc2
Description	Stable 293T-NFAT-Luc2 cell line expressing GNRHR and exogenous luciferase under the control of NFAT signaling pathway.
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	70% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 150μg/mL Hygromycin B + 1μg/mL Puromycin
Selection Marker	Hygromycin B, Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1 : 10 every 3 days; seed out at about 1 × 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-NFAT-Luc2-GNRHR Cell Line was generated using a lentiviral vector expressing the GNRHR sequence.

Characterization

Figure1: 293T-NFAT-Luc2-GNRHR cells were seeded into 96-well plates, treated with LH-RH for 16 hours, and then read out using Bright-Glo Detection System.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS, 150μg/mL Hygromycin B and 1μg/mL Puromycin.) 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 : 10 every 3 days; seed out at about 1 × 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

1. Kottler ML, Lorenzo F, Bergametti F, Commerçon P, Souchier C, Counis R. Subregional mapping of the human gonadotropin-releasing hormone receptor (GnRH-R) gene to 4q between the markers D4S392 and D4S409. Hum Genet. 1995 Oct;96(4):477-80. doi: 10.1007/BF00191810. PMID: 7557974.
2. Trarbach EB, Silveira LG, Latronico AC. Genetic insights into human isolated gonadotropin deficiency. Pituitary. 2007;10(4):381-91. doi: 10.1007/s11102-007-0061-7. PMID: 17624596.