KC-3615

293T-CRE-Luc2-FSHR Cell Line

34550

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Background of 293T-CRE-Luc2-FSHR Cell Line

Cyclic adenosine monophosphate (cAMP) is a second messenger involved in cell signaling that regulates variousl physiological and pathological processes. cAMP regulates the transcription of target genes by activating proteinl kinase A (PKA) and the transcription factor cAMP response element-binding protein (CREB). CRE is the target of many extracellular and intracellular signaling pathways, including cAMP, calcium,l GPCR (G-protein coupled receptors), and neurotrophins. The CAMP/PKA/CREB signaling pathway has both tumor-suppressive and tumor-promoting effects in cancer cells and can be useful in studying cancer signaling pathways. Follicle-stimulating hormone (FSH) is synthesized by the anterior pituitary gonadotroph cells, and it plays a critical role in controlling male and female gonadal function . FSH acts through its specific receptor (FSHR), a member of the highly conserved family of class A G-protein-coupled receptors (GPCR). It has a long ECD, a 7 transmembrane domain, 3 short intracellular loops, 3 extra loops and an intracellular tail. FSHR binds to FSH by the very large ECD. Multiple isoforms of FSHR have been reported and FSHR has been also expressed on extragonadal tissues including placenta, uterus, prostate, bone tissue and ovarian epithelium as well as ovarian cancer. FSHR has also been found to be selectively expressed on the surface of many tumor blood vessels, and related to tumor metastasis. FSHR activation may trigger a number of intracellular signaling pathways that will be activated in parallel or sequentially. The canonical Gsα/cAMP/PKA signaling pathway, a key effector mechanism of FSH action, activates the cAMP response element-binding protein that modulates gene transcription. Corifollitropin alfa (Elonva®) is a fusion product of human follicle-stimulating hormone (FSH) and the C-terminal peptide of the β-subunit of human chorionic gonadotropin (hCG) produced by recombinant DNA technology. It has the same pharmacologic activity as FSH and recombinant FSH (rFSH; follitropin alfa; follitropin beta), but with a slower absorption and a longer half-life.

Specifications

Catalog Number	KC-3615
Cell Line Name	293T-CRE-Luc2-FSHR Cell Line
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous luciferase gene under the control of FSHR signaling pathway
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Cell model for monitoring FSHR signaling pathway
Freezing Medium	70% DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10% FBS +150μg/mL Hygromycin+1μg/mL Puromycin
Selection Marker	Hygromycin and Puromycin
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-CRE-Luc2-FSHR cell line was generated using lentivirus expressing FSHR sequence.

Characterization

Figure 1. 293T-CRE-Luc2-FSHR cell line was seeded into the 96-well plate, and treated with Elonva at a maximum concentration 100ng/mL incubated for 16 hours and readout with Bright-Glo system.

Figure2. 293T-CRE-Luc2-FSHR cells were seeded into 96-well plates, treated with FSHα/β for 16 hours, and then read out using Bright-Glo Detection System.

Figure2. 293T-CRE-Luc2-FSHR cells were seeded into 96-well plates, treated with HuF13 and 9H11 for 1 hours, and then treated with FSHα/β in the concentration of 1ng/mL 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 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 1:4-1:8 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

Szymańska K, Kałafut J, Przybyszewska A, Paziewska B, Adamczuk G, Kiełbus M, Rivero-Müller A. FSHR Trans-Activation and Oligomerization. Front Endocrinol (Lausanne). 2018 Dec 13;9:760. doi: 10.3389/fendo.2018.00760. PMID: 30619090; PMCID: PMC6301190.
Bhartiya D, Patel H. An overview of FSH-FSHR biology and explaining the existing conundrums. J Ovarian Res. 2021 Oct 30;14(1):144. doi: 10.1186/s13048-021-00880-3. PMID: 34717708; PMCID: PMC8557046.
Croxtall JD, McKeage K. Corifollitropin alfa: a review of its use in controlled ovarian stimulation for assisted reproduction. BioDrugs. 2011 Aug 1;25(4):243-54. doi: 10.2165/11206890-000000000-00000. PMID: 21815699.
Chrusciel M, Ponikwicka-Tyszko D, Wolczynski S, Huhtaniemi I, Rahman NA. Extragonadal FSHR Expression and Function-Is It Real? Front Endocrinol (Lausanne). 2019 Feb 4;10:32. doi: 10.3389/fendo.2019.00032. PMID: 30778333; PMCID: PMC6369633.