KC-3779

293T-mouse-LGR4 Cell Line

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Background of 293T-mouse-LGR4 Cell Line

Enables transmembrane signaling receptor activity. Involved in several processes, including metanephric nephron development; regulation of gene expression; and regulation of signal transduction. Acts upstream of or within several processes, including epithelial cell proliferation involved in renal tubule morphogenesis; intestinal stem cell homeostasis; and male genitalia development. Located in plasma membrane. Is expressed in several structures, including alimentary system; eye; integumental system; metanephros; and telencephalon. Used to study glaucoma. Orthologous to human LGR4 (leucine rich repeat containing G protein-coupled receptor 4).

Specifications

Catalog Number	KC-3779
Cell Line Name	293T-mouse-LGR4 Cell Line
Host Cell Line	293T
Description	Stable HEK293T clone expressing exogenous mouse LGR4 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 1:4-1:5 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 mouse LGR4 cell line was generated using a lentiviral vector expressing the mouse LGR4 sequence.

Characterization

Figure 1: Characterization of mouse LGR4 overexpression in the 293T mouse LGR4 stable clone using qPCR.

Figure 2: Characterization of mouse LGR4 in the 293T mouse LGR4 stable clone 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 1:4-1:5 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. Liang Y, Luo C, Sun L, Feng T, Yin W, Zhang Y, Mulholland MW, Zhang W, Yin Y. Reduction of specific enterocytes from loss of intestinal LGR4 improves lipid metabolism in mice. Nat Commun. 2024 May 23;15(1):4393. doi: 10.1038/s41467-024-48622-5. PMID: 38782937; PMCID: PMC11116434. 2. Guan X, Duan Y, Zeng Q, Pan H, Qian Y, Li D, Cao X, Liu M. Lgr4 protein deficiency induces ataxia-like phenotype in mice and impairs long term depression at cerebellar parallel fiber-Purkinje cell synapses. J Biol Chem. 2014 Sep 19;289(38):26492-26504. doi: 10.1074/jbc.M114.564138. Epub 2014 Jul 25. PMID: 25063812; PMCID: PMC4176234.