KC-4490

293T-LRP6-beta-propeller-3-4-MESD-Middle-Cell-Line

46359

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Background of 293T-LRP6-beta-propeller-3-4-MESD-Middle-Cell-Line

This gene encodes a member of the low density lipoprotein (LDL) receptor gene family. LDL receptors are transmembrane cell surface proteins involved in receptor-mediated endocytosis of lipoprotein and protein ligands. The protein encoded by this gene functions as a receptor or, with Frizzled, a co-receptor for Wnt and thereby transmits the canonical Wnt/beta-catenin signaling cascade. Through its interaction with the Wnt/beta-catenin signaling cascade this gene plays a role in the regulation of cell differentiation, proliferation, and migration and the development of many cancer types. This protein undergoes gamma-secretase dependent RIP- (regulated intramembrane proteolysis) processing but the precise locations of the cleavage sites have not been determined.

Specifications

Catalog Number	KC-4490
Cell Line Name	293T-LRP6-beta-propeller-3-4-MESD-Middle-Cell-Line
Host Cell Line	293T
Description	Stable HEK293T clone expressing exogenous human LRP6 gene in middle level
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-LRP6-beta-propeller-3-4-MESD-Middle cell line was generated using a lentiviral vector expressing the human LRP6 sequence.

Characterization

Figure 1: Characterization of human LRP6 overexpression in the 293T-LRP6-beta-propeller-3-4-MESD stable clone using FACS.

Figure 2: Characterization of human LRP6 in the 293T-LRP6-beta-propeller-3-4-MESD stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS 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 1:4-1:5 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

Xu Y, Gong W, Peng J, Wang H, Huang J, Ding H, Wang DW. Functional analysis LRP6 novel mutations in patients with coronary artery disease. PLoS One. 2014 Jan 10;9(1):e84345.
Singh R, Smith E, Fathzadeh M, Liu W, Go GW, Subrahmanyan L, Faramarzi S, McKenna W, Mani A. Rare nonconservative LRP6 mutations are associated with metabolic syndrome. Hum Mutat. 2013 Sep;34(9):1221-5.