KC-5328

SHP77-DLL3-SEZ6-KO Cell Line

57028

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Background of SHP77-DLL3-SEZ6-KO Cell Line

The seizure-related 6 homolog (SEZ6) gene encodes a transmembrane protein predominantly expressed in the central nervous system, with emerging roles in neuronal development and synaptic function. SEZ6 regulates dendritic arborization and modulates excitatory synaptic transmission through interactions with postsynaptic density proteins. Genome-wide association studies implicate SEZ6 in neuropsychiatric disorders, including autism spectrum disorder and schizophrenia, where its dysregulation may contribute to synaptic dysfunction. Recent studies also identify SEZ6 as a substrate for β-secretase (BACE1), linking it to amyloid precursor protein processing in Alzheimer's disease. While SEZ6 knockout mice show behavioral abnormalities and altered seizure thresholds, the precise molecular mechanisms remain unclear. Further investigation of SEZ6's role in neural circuit formation and its potential as a therapeutic target for neurological disorders is warranted.

Specifications

Catalog Number	KC-5328
Cell Line Name	SHP77-DLL3-SEZ6-KO Cell Line
Clone Number	1D3
Host Cell Line	SHP77-DLL3-KO
Description	Stable SHP77-DLL3-KO clone with human SEZ6 gene knockout
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
Selection Marker	N/A
Morphology	rounded
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

SHP77-DLL3-SEZ6-KO cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of SHP77-DLL3-SEZ6-KO cell line stable clone using PCR sequencing.

Figure 2: Characterization of SHP77-DLL3-SEZ6-KO cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of SHP77 (Left) and SHP77-DLL3-SEZ6-KO (Right) Cell Lines stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (RPMI1640+10% FBS)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% 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

Gunnersen JM, et al. (2007). "Sez-6 proteins affect dendritic arborization patterns and excitability of cortical pyramidal neurons." Neuron, 56(4):621-39.
Hoshino F, et al. (2020). "SEZ6 regulates excitatory synapse density in cortical neurons." Molecular Brain, 13(1):103.
Bellingham M, et al. (2022). "The BACE1 substrate SEZ6 is a regulator of excitatory synapses." Journal of Neuroscience, 42(18):3731-3745.