KC-3569

CHOK1-chimeric-DLL3-EGF1-Cell-Line

26411

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Background of CHOK1-chimeric-DLL3-EGF1-Cell-Line

DLL3 (Delta Like Canonical Notch Ligand 3), is also known as SCDO1. It is a member of ligand protein family, functioned as Notch ligand, and play a important role in somitogensis. Mutations in this gene cause autosomal recessive spondylocostal dysostosis 1. DLL3 is also overexpression in neuroendocrine tumors, which has been identified as a potential target for tumor therapy. Rovapituzumab tesirine, which targeted DLL3, have been used as experimental treatment of lung cancer. Egf1 fibropellin I also known as EGF1. EGF1 peptide may be a good target head for delivering drugs to TF in anticoagulation therapy.

Specifications

Catalog Number	KC-3569
Cell Line Name	CHOK1-chimeric-DLL3-EGF1-Cell-Line
Host Cell Line	CHOK1
Description	Stable CHOK1 cell line expressing exogenous chimeric-DLL3-EGF1 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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS + 10μg/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 24 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

CHOK1-chimeric-DLL3-EGF1 cell line was generated using a lentiviral vector expressing the chimeric-DLL3-EGF1 sequence.

Characterization

Figure 1: Characterization of chimeric-DLL3-EGF1 overexpression in CHOK1 stable clones using FACS..

Figure 2: Characterization of chimeric-DLL3-EGF1 and its mutants overexpressing in CHOK1 stable clones using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 10μg/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

Matsuo K, Taniguchi K, Hamamoto H, Inomata Y, Komura K, Tanaka T, Lee SW, Uchiyama K. Delta-like canonical Notch ligand 3 as a potential therapeutic target in malignancies: A brief overview. Cancer Sci. 2021 Aug;112(8):2984-2992. doi: 10.1111/cas.15017. Epub 2021 Jun 30. PMID: 34107132; PMCID: PMC8353941.
Yao J, Bergsland E, Aggarwal R, Aparicio A, Beltran H, Crabtree JS, Hann CL, Ibrahim T, Byers LA, Sasano H, Umejiego J, Pavel M. DLL3 as an Emerging Target for the Treatment of Neuroendocrine Neoplasms. Oncologist. 2022 Nov 3;27(11):940-951. doi: 10.1093/oncolo/oyac161. PMID: 35983951; PMCID: PMC9632312.
Mei H, Hu Y, Wang H, Shi W, Deng J, Guo T. Binding of EGF1 domain peptide in coagulation factor VII with tissue factor and its implications for the triggering of coagulation. J Huazhong Univ Sci Technolog Med Sci. 2010 Feb;30(1):42-7. doi: 10.1007/s11596-010-0108-2. Epub 2010 Feb 14. PMID: 20155454.