KC-4552

CHOK1-Tet-on-MAPT-P301L-low Cell Line

61206

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Background of CHOK1-Tet-on-MAPT-P301L-low Cell Line

MAPT (Microtubule Associated Protein Tau) is a Protein Coding gene. This gene encodes the microtubule-associated protein tau (MAPT) whose transcript undergoes complex, regulated alternative splicing, giving rise to several mRNA species. MAPT transcripts are differentially expressed in the nervous system, depending on stage of neuronal maturation and neuron type. MAPT gene mutations have been associated with several neurodegenerative disorders such as Alzheimer's disease, Pick's disease, frontotemporal dementia, cortico-basal degeneration and progressive supranuclear palsy.

Specifications

Catalog Number	KC-4552
Cell Line Name	CHOK1-Tet-on-MAPT-P301L-low Cell Line
NCBI/UniProt Accession Number	NM_005910.6
Clone Number	2#
Host Cell Line	CHOK1
Description	CHOK1 cell line stably expressing exogenous MAPT-P301L gene, The gene is under the control of a tet-on inducible system.
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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS + 10 μg/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

CHOK1-Tet-on-MAPT-P301L-low cell line was generated using a lentiviral vector expressing the MAPT-P301L sequence, MAPT-P301L gene is under the control of a tet-on inducible system.

Characterization

Figure1: Characterization of MAPT-P301L overexpressing in CHOK1 stable clones using WB (clone 2#).

Figure2: Characterization of CHOK1-Tet-on-MAPT-P301L-low cell line stable clone using PCR sequencing (clone 2#).

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS + 10 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 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 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

Basurto-Islas G, Luna-Muñoz J, Guillozet-Bongaarts AL, Binder LI, Mena R, García-Sierra F. Accumulation of aspartic acid421- and glutamic acid391-cleaved tau in neurofibrillary tangles correlates with progression in Alzheimer disease. J Neuropathol Exp Neurol. 2008 May;67(5):470-83. doi: 10.1097/NEN.0b013e31817275c7. PMID: 18431250; PMCID: PMC4699801.
Djukic M, Spreer A, Lange P, Bunkowski S, Wiltfang J, Nau R. Small cisterno-lumbar gradient of phosphorylated Tau protein in geriatric patients with suspected normal pressure hydrocephalus. Fluids Barriers CNS. 2016 Aug 31;13(1):15. doi: 10.1186/s12987-016-0039-9. PMID: 27581842; PMCID: PMC5007695.
Silva MC, Cheng C, Mair W, Almeida S, Fong H, Biswas MHU, Zhang Z, Huang Y, Temple S, Coppola G, Geschwind DH, Karydas A, Miller BL, Kosik KS, Gao FB, Steen JA, Haggarty SJ. Human iPSC-Derived Neuronal Model of Tau-A152T Frontotemporal Dementia Reveals Tau-Mediated Mechanisms of Neuronal Vulnerability. Stem Cell Reports. 2016 Sep 13;7(3):325-340. doi: 10.1016/j.stemcr.2016.08.001. Epub 2016 Sep 1. PMID: 27594585; PMCID: PMC5032560.