KC-4106

293T-CD46-KO-2A2-Cell-Line

41492

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Background of 293T-CD46-KO-2A2-Cell-Line

CD46 is a ubiquitously expressed type I transmembrane protein, first identified as a regulator of complement activation, and later as an entry receptor for a variety of pathogens. The last decade has also revealed the role of CD46 in regulating the adaptive immune response, acting as an additional costimulatory molecule for human T cells and inducing their differentiation into Tr1 cells, a subset of regulatory T cells. Interestingly, CD46 regulatory pathways are defective in T cells from patients with multiple sclerosis, asthma and rheumatoid arthritis, illustrating its importance in regulating T cell homeostasis. Indeed, CD46 expression at the cell surface is tightly regulated in many different cell types, highlighting its importance in several biological processes. Notably, CD46 is the target of enzymatic processing, being cleaved by metalloproteinases and by the presenilin/gamma secretase complex. This processing is required for its functions, at least in T cells.

Specifications

Catalog Number	KC-4106
Cell Line Name	293T-CD46-KO-2A2-Cell-Line
Host Cell Line	293T
Description	Stable 293T clone with human CD46 gene knockout, No.2A2
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	DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10% FBS
Selection Marker	N/A
Morphology	Epithelial
Subculture	Split saturated culture 1:5-1:10 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-CD46-KO-2A2 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of 293T-CD46-KO-2A2 cell line stable clone using PCR sequencing.

Figure 2: Characterization of293T-CD46-KO-2A2 cell line stable clone using FACS.

Cell Resuscitation

"1. Prewarm culture medium (DMEM + 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 1:5-1:10 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.
"

Cell Freezing

"1. 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

Ni Choileain S, Astier AL. CD46 processing: a means of expression. Immunobiology. 2012 Feb;217(2):169-75. doi: 10.1016/j.imbio.2011.06.003. Epub 2011 Jul 13. PMID: 21742405; PMCID: PMC4363545.