KC-4013

293T-ACVR2A-ACVR2B-KO-2C2 Cell Line

40484

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Background of 293T-ACVR2A-ACVR2B-KO-2C2 Cell Line

"ACVR2A gene also called ACVR2, ACTRII, or ACTRIIA is located on the long arm of chromosome 2 (location 2q22.2-q23.3) and has the overall length of 83.3 kb. The lead role of a protein encoded by this gene is the mediation of activin functions. The activin A receptor type 2A (ACVR2A) is constructed of 513 amino acids, and consists of an extracellular, a transmembrane and a cytoplasmic serine-threonine kinase domains. Except for the ability to transfer phosphate groups, the protein exhibits transferase and tyrosine kinase activity. ACVR2B is a single‐transmembrane‐domain serine/threonine kinase receptor that acts as a type II receptor; it initiates signaling and cellular responses through binding to ligands, such as GDF5, 8, 11, and activin A. The downstream function depends on which binding partner engages the receptor. Transmembrane serine/threonine kinase activin type-2 receptor forming an activin receptor complex with activin type-1 serine/threonine kinase receptors (ACVR1, ACVR1B or ACVR1c). Transduces the activin signal from the cell surface to the cytoplasm and is thus regulating many physiological and pathological processes including neuronal differentiation and neuronal survival, hair follicle development and cycling, FSH production by the pituitary gland, wound healing, extracellular matrix production, immunosuppression and carcinogenesis. "

Specifications

Catalog Number	KC-4013
Cell Line Name	293T-ACVR2A-ACVR2B-KO-2C2 Cell Line
Host Cell Line	293T
Description	Stable 293T clone with human ACVR2A and ACVR2B 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	DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10% FBS
Selection Marker	NA
Morphology	epithelial
Subculture	Split saturated culture 1:5-1:8 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 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

293T-ACVR2A-ACVR2B-KO-2C2 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of 293T-ACVR2A-ACVR2B-KO cell line stable clone using PCR sequencing.

Figure 2: Characterization of 293T-ACVR2A-ACVR2B-KO cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of 293T-ACVR2A-ACVR2B-KO cell line stable clone using FACS.

Cell Resuscitation

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 1:5-1:8 every 2-3 days; seed out at about 1-3 ×10⁵ cells/mL.

Cell Freezing

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

Wodziński D, Wosiak A, Pietrzak J, Świechowski R, Jeleń A, Balcerczak E. Does the expression of the ACVR2A gene affect the development of colorectal cancer? Genet Mol Biol. 2019 Jan-Mar;42(1):32-39.
Jung B, Doctolero RT, Tajima A, Nguyen AK, Keku T, Sandler RS, Carethers JM. Loss of Activin Receptor Type 2 protein expression in microsatellite unstable colon cancers. Gastroenterology. 2004;126:654–659.
Jeon J, Lee H, Jeon MS, Kim SJ, Choi C, Kim KW, Yang DJ, Lee S, Bae YS, Choi WI, Jung J, Eyun SI, Yang S. Blockade of Activin Receptor IIB Protects Arthritis Pathogenesis by Non-Amplification of Activin A-ACVR2B-NOX4 Axis Pathway. Adv Sci (Weinh). 2023 May;10(14):e2205161. doi: 10.1002/advs.202205161. Epub 2023 Mar 22. PMID: 36950748; PMCID: PMC10190289.