KC-3855

293T-LRRK2-G2019S-Cell-Line

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Background of 293T-LRRK2-G2019S-Cell-Line

LRRK2 is a leucine-rich repeat-rich kinase 2 whose mutations are commonly implicated in the pathogenesis of familial and sporadic Parkinson's disease (PD). LRRK2 regulates key cellular processes at membrane organelles and forms microtubo-based pathogenic microfilaments. LRRK2 protein is also associated with autophagic pathways and lysosomal activity in neurons.

Specifications

Catalog Number	KC-3855
Cell Line Name	293T-LRRK2-G2019S-Cell-Line
Host Cell Line	293T
Description	Stable HEK293T clone expressing exogenous human LRRK2 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
In Vivo Validation	NA

Cell Line Generation

293T LRRK2 G2019S cell line was generated using a lentiviral vector expressing the human LRRK2 sequence.

Characterization

Figure 1: Characterization of human LRRK2 overexpression in the 293T human LRRK2 G2019S stable clone using qPCR.

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 1:4-1:8 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

Madureira M, Connor-Robson N, Wade-Martins R. LRRK2: Autophagy and Lysosomal Activity. Front Neurosci. 2020 May 25;14:498. doi: 10.3389/fnins.2020.00498. PMID: 32523507; PMCID: PMC7262160.
Chang EES, Ho PW, Liu HF, Pang SY, Leung CT, Malki Y, Choi ZY, Ramsden DB, Ho SL. LRRK2 mutant knock-in mouse models: therapeutic relevance in Parkinson's disease. Transl Neurodegener. 2022 Feb 14;11(1):10. doi: 10.1186/s40035-022-00285-2. PMID: 35152914; PMCID: PMC8842874.
Chang EES, Ho PW, Liu HF, Pang SY, Leung CT, Malki Y, Choi ZY, Ramsden DB, Ho SL. LRRK2 mutant knock-in mouse models: therapeutic relevance in Parkinson's disease. Transl Neurodegener. 2022 Feb 14;11(1):10. doi: 10.1186/s40035-022-00285-2. PMID: 35152914; PMCID: PMC8842874.