KC-5641

293T-cyno-LGR5 Cell Line

57651

Home » 293T-cyno-LGR5 Cell Line

Background of 293T-cyno-LGR5 Cell Line

LGR5 (Leucine Rich Repeat Containing G Protein-Coupled Receptor 5) is a leucine-rich repeat-containing receptor (LGR) and member of the G protein-coupled, 7-transmembrane receptor (GPCR) superfamily. LGR5 is a marker of normal and cancer stem cells in various tissues where it functions as a receptor for R-spondins and increases canonical Wnt signalling amplitude. Diseases associated with LGR5 include Ankyloglossia With Or Without Tooth Anomalies and Mesenteric Vascular Occlusion.

Specifications

Catalog Number	KC-5641
Cell Line Name	293T-cyno-LGR5 Cell Line
Clone Number	7#
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous cyno LGR5 gene
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 + 1μg/mL Puromycin
Selection Marker	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

Cell Line Generation

293T-cyno-LGR5 cell line was generated using a lentiviral vector expressing the cyno LGR5 sequence.

Characterization

Figure 1: Characterization of LGR5 overexpression in the 293T cyno LGR5 stable clone using FACS.

Figure 2: Characterization of cyno LGR5 and its mutants overexpressing in 293T stable clones using PCR sequencing.

Cell Resuscitation

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

Cell Freezing

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

References

1. Vieira GC, Chockalingam S, Melegh Z, Greenhough A, Malik S, Szemes M, Park JH, Kaidi A, Zhou L, Catchpoole D, Morgan R, Bates DO, Gabb PD, Malik K. LGR5 regulates pro-survival MEK/ERK and proliferative Wnt/β-catenin signalling in neuroblastoma. Oncotarget. 2015 Nov 24;6(37):40053-67. doi: 10.18632/oncotarget.5548. Erratum in: Oncotarget. 2017 May 9;8(19):32381. doi: 10.18632/oncotarget.17685. PMID: 26517508; PMCID: PMC4741879.
2. Miao J, Jiang Y, Wang D, Zhou J, Fan C, Jiao F, Liu B, Zhang J, Wang Y, Zhang Q. Trichosanthin suppresses the proliferation of glioma cells by inhibiting LGR5 expression and the Wnt/β-catenin signaling pathway. Oncol Rep. 2015 Dec;34(6):2845-52. doi: 10.3892/or.2015.4290. Epub 2015 Sep 18. PMID: 26397053; PMCID: PMC4722885.
3. Wang Z, Liu C. Lgr5-Positive Cells are Cancer-Stem-Cell-Like Cells in Gastric Cancer. Cell Physiol Biochem. 2015;36(6):2447-55. doi: 10.1159/000430205. Epub 2015 Jul 27. PMID: 26279446.