KC-6391

MDA-MB-468-PVRL1-low Cell Line

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Background of MDA-MB-468-PVRL1-low Cell Line

PVRL1 aliases is NECTIN1. NECTIN1 (Nectin Cell Adhesion Molecule 1) is a Protein Coding gene. This gene encodes an adhesion protein that plays a role in the organization of adherens junctions and tight junctions in epithelial and endothelial cells. The protein is a calcium(2+)-independent cell-cell adhesion molecule that belongs to the immunoglobulin superfamily and has 3 extracellular immunoglobulin-like loops, a single transmembrane domain (in some isoforms), and a cytoplasmic region. This protein acts as a receptor for glycoprotein D (gD) of herpes simplex viruses 1 and 2 (HSV-1, HSV-2), and pseudorabies virus (PRV) and mediates viral entry into epithelial and neuronal cells. Mutations in this gene cause cleft lip and palate/ectodermal dysplasia 1 syndrome (CLPED1) as well as non-syndromic cleft lip with or without cleft palate (CL/P). Alternative splicing results in multiple transcript variants encoding proteins with distinct C-termini.

Specifications

Catalog Number	KC-6391
Cell Line Name	MDA-MB-468-PVRL1-low Cell Line
NCBI/UniProt Accession Number	NM_002855.5
Clone Number	1#
Host Cell Line	MDA-MB-468
Description	MDA-MB-468 cell line stably expressing exogenous PVRL1 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 + 1 µg/mL puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:2-1:4 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

MDA-MB-468-PVRL1-low cell line was generated using a transposon vector expressing the PVRL1 sequence.

Characterization

Figure1: Characterization of PVRL1 overexpressing in MDA-MB-468-PVRL1-low stable clone using FACS.

Figure2: Characterization of MDA-MB-468-PVRL1-low cell line stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS + 1 µg/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 1:2-1:4 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

Tiwari V, Oh MJ, Kovacs M, Shukla SY, Valyi-Nagy T, Shukla D. Role for nectin-1 in herpes simplex virus 1 entry and spread in human retinal pigment epithelial cells. FEBS J. 2008 Nov;275(21):5272-85. doi: 10.1111/j.1742-4658.2008.06655.x. Epub 2008 Sep 17. PMID: 18803666; PMCID: PMC2758088.
Oner DA, Tastan H. Identification of Novel Variants in the PVRL1 Gene in Patients With Nonsyndromic Cleft Lip With or Without Cleft Palate. Genet Test Mol Biomarkers. 2016 May;20(5):269-72. doi: 10.1089/gtmb.2015.0276. Epub 2016 Mar 8. PMID: 26953873.