KC-5487

293T-cyno-LRP5-D1/2 Cell Line

57482

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Background of 293T-cyno-LRP5-D1/2 Cell Line

This gene encodes a transmembrane low-density lipoprotein receptor that binds and internalizes ligands in the process of receptor-mediated endocytosis. This protein also acts as a co-receptor with Frizzled protein family members for transducing signals by Wnt proteins and was originally cloned on the basis of its association with type 1 diabetes mellitus in humans. This protein plays a key role in skeletal homeostasis and many bone density related diseases are caused by mutations in this gene. Mutations in this gene also cause familial exudative vitreoretinopathy. Alternative splicing results in multiple transcript variants.

Specifications

Catalog Number	KC-5487
Cell Line Name	293T-cyno-LRP5-D1/2 Cell Line
NCBI/UniProt Accession Number	XM_045370597.1
Clone Number	9-10#
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous cyno-LRP5-D1/2 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-LRP5-D1/2 cell line was generated using lentivirus expressing cyno-LRP5-D1/2 sequence.

Characterization

Figure 1. Characterization of cyno-LRP5-D1/2 over-expression in the 293T-cyno-LRP5-D1/2 stable clone using FACS.(Primary antibody: F013500571-3-hIgG1m29-FC, Cat#KB-1464, Kyinno)

Figure 2. Characterization of cyno-LRP5-D1/2 over-expression in the 293T-cyno-LRP5-D1/2 stable clone using PCR.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS, 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

Panach L, Mifsut D, Tarín JJ, Cano A, García-Pérez MÁ. Replication study of three functional polymorphisms associated with bone mineral density in a cohort of Spanish women. J Bone Miner Metab. 2014 Nov;32(6):691-8. doi: 10.1007/s00774-013-0539-5. Epub 2013 Dec 14. PMID: 24337955.
Xu GY, Qiu Y, Mao HJ. Common polymorphism in the LRP5 gene may increase the risk of bone fracture and osteoporosis. Biomed Res Int. 2014;2014:290531. doi: 10.1155/2014/290531. Epub 2014 Dec 14. PMID: 25580429; PMCID: PMC4279179.