KC-5542

CHOK1-CTLA4-PD1 Cell Line

60104

Home » CHOK1-CTLA4-PD1 Cell Line

Background of CHOK1-CTLA4-PD1 Cell Line

Despite the knowledge of many genetic alterations present in osteosarcoma, the complexity of this disease precludes placing its biology into a simple conceptual framework. Cytotoxic T-lymphocyte antigen-4 (CTLA-4) plays important roles in downregulating T-cell activation, thereby attenuating anti-tumor responses and increasing cancer susceptibility. Polymorphisms in the CTLA-4 gene are associated with different autoimmune diseases and cancers. Plastid-encoded RNA polymerase (PEP)-dependent transcription is an essential process for chloroplast development and plant growth. It is a complex event that is regulated by numerous nuclear-encoded proteins. In order to elucidate the complex regulation mechanism of PEP activity, identification and characterization of PEP activity regulation factors are needed. Here, we characterize Plastid Deficient 1 (PD1) as a novel regulator for PEP-dependent gene expression and chloroplast development in Arabidopsis. The PD1 gene encodes a protein that is conserved in photoautotrophic organisms. The Arabidopsis pd1 mutant showed albino and seedling-lethal phenotypes. The plastid development in the pd1 mutant was arrested. The PD1 protein localized in the chloroplasts, and it colocalized with nucleoid protein TRXz.

Specifications

Catalog Number	KC-5542
Cell Line Name	CHOK1-CTLA4-PD1 Cell Line
NCBI/UniProt Accession Number	NM_005214.5, NM_005018.3
Clone Number	18#
Host Cell Line	CHOK1
Description	CHOK1 cell line stably expressing exogenous CTLA4 and PD1 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 + 10 μg/mL BSD
Selection Marker	Blasticidin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-2 × 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-CTLA4-PD1 cell line was generated using a lentiviral vector expressing the CTLA4 and PD1 sequence.

Characterization

Figure 1: Characterization of CTLA1-PD1 overexpression in CHOK1-CTLA1-PD1 stable clones using FACS.

Figure 2:Characterization of CHOK1-CTLA1-PD1 cell line stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 10 μg/mL BSD) 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 9.0 mL 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 1-2 × 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

Liu Y, He Z, Feng D, Shi G, Gao R, Wu X, Song W, Yuan W. Cytotoxic T-lymphocyte antigen-4 polymorphisms and susceptibility to osteosarcoma. DNA Cell Biol. 2011 Dec;30(12):1051-5. doi: 10.1089/dna.2011.1269. Epub 2011 May 25. PMID: 21612409.
Yang Z, Liu M, Ding S, Zhang Y, Yang H, Wen X, Chi W, Lu C, Lu Q. Plastid Deficient 1 Is Essential for the Accumulation of Plastid-Encoded RNA Polymerase Core Subunit β and Chloroplast Development in Arabidopsis. Int J Mol Sci. 2021 Dec 20;22(24):13648. doi: 10.3390/ijms222413648. PMID: 34948448; PMCID: PMC8705867.