KC-4072

H4-GBA1-KO Cell Line

40602

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Background of H4-GBA1-KO Cell Line

The GBA1 gene, located on chromosome 1q21, encodes the lysosomal enzyme glucocerebrosidase (GCase), which is crucial for the breakdown of glucocerebroside into glucose and ceramide. Mutations in GBA1 are most commonly associated with Gaucher disease, a lysosomal storage disorder characterized by the accumulation of glucocerebroside in macrophages, leading to hepatosplenomegaly, anemia, and bone abnormalities (Sidransky et al., 2009). Beyond Gaucher disease, GBA1 mutations are a significant genetic risk factor for Parkinson's disease (PD) and related synucleinopathies. Studies suggest that impaired GCase activity contributes to the accumulation of alpha-synuclein, a hallmark of PD pathology (Alcalay et al., 2012). The GBA1 gene has over 400 known mutations, with varying impacts on enzyme activity and disease severity. Research continues to explore the mechanisms linking GBA1 dysfunction to neurodegenerative diseases, offering potential therapeutic targets.

Specifications

Catalog Number	KC-4072
Cell Line Name	H4-GBA1-KO Cell Line
Host Cell Line	H4
Description	Stable H4 clone with human GBA1 gene knockout, No.1A2
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	RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10% FBS+GlutaMAX
Selection Marker	NA
Morphology	epithelial
Subculture	Split saturated culture 1:5 every 2 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 23 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

H4-GBA1-KO cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of H4-GBA1-KO cell line stable clone using PCR sequencing.

Figure 2: Characterization of H4-GBA1-KO cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of H4-GBA1-KO cell line stable clone using western blot.

Cell Resuscitation

Prewarm culture medium (RPMI1640+10% FBS+GlutaMAX) 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 1:5 every 2 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI-1640 + 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

Sidransky E, Nalls MA, Aasly JO, Aharon-Peretz J, Annesi G, Barbosa ER, Bar-Shira A, Berg D, Bras J, Brice A, Chen CM, Clark LN, Condroyer C, De Marco EV, Dürr A, Eblan MJ, Fahn S, Farrer MJ, Fung HC, Gan-Or Z, Gasser T, Gershoni-Baruch R, Giladi N, Griffith A, Gurevich T, Januario C, Kropp P, Lang AE, Lee-Chen GJ, Lesage S, Marder K, Mata IF, Mirelman A, Mitsui J, Mizuta I, Nicoletti G, Oliveira C, Ottman R, Orr-Urtreger A, Pereira LV, Quattrone A, Rogaeva E, Rolfs A, Rosenbaum H, Rozenberg R, Samii A, Samaddar T, Schulte C, Sharma M, Singleton A, Spitz M, Tan EK, Tayebi N, Toda T, Troiano AR, Tsuji S, Wittstock M, Wolfsberg TG, Wu YR, Zabetian CP, Zhao Y, Ziegler SG. Multicenter analysis of glucocerebrosidase mutations in Parkinson's disease. N Engl J Med. 2009 Oct 22;361(17):1651-61. doi: 10.1056/NEJMoa0901281. PMID: 19846850; PMCID: PMC2856322.
Alcalay RN, Levy OA, Waters CC, Fahn S, Ford B, Kuo SH, Mazzoni P, Pauciulo MW, Nichols WC, Gan-Or Z, Rouleau GA, Chung WK, Wolf P, Oliva P, Keutzer J, Marder K, Zhang X. Glucocerebrosidase activity in Parkinson's disease with and without GBA mutations. Brain. 2015 Sep;138(Pt 9):2648-58. doi: 10.1093/brain/awv179. Epub 2015 Jun 27. PMID: 26117366; PMCID: PMC4564023.