KC-4923

293T-SMAD-Luc2-ACVR2A-KO Cell Line

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Background of 293T-SMAD-Luc2-ACVR2A-KO 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. "

Specifications

Catalog Number	KC-4923
Cell Line Name	293T-SMAD-Luc2-ACVR2A-KO Cell Line
Clone Number	1B3
Host Cell Line	293T-SMAD-Luc2
Description	Stable 293T-SMAD-Luc2 clone with human ACVR2A gene knockout, No.1B3
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+150μg/mL HygromycinB
Selection Marker	Hygromycin B
Morphology	epithelial
Subculture	Split saturated culture 1:5-1:8 every 2-3 days; seed out at about 1 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 40 hours
Mycoplasma Status	Negative

Cell Line Generation

293T-SMAD-Luc2-ACVR2A-KO cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of 293T-SMAD-Luc2-ACVR2A-KO cell line stable clone using PCR sequencing.

Figure 2: Characterization of 293T-SMAD-Luc2-ACVR2A-KO cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of 293T-SMAD-Luc2-ACVR2A-KO cell line stable clone using FACS.

Figure 4: 293T-SMAD-Luc2-ACVR2A-KO cells were seeded into 96-well plates, treated with Activin A for 16 hours, and then read out using Bright-Glo Detection System.

Figure 5: 293T-SMAD-Luc2-ACVR2A-KO cells were seeded into 96-well plates, treated with Bimagrumab (Cat# KB-1390, Kyinno) for 1 hours, and then treated with 50ng/mL Activin A for 16 hours, and then read out using Bright-Glo Detection System.

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS+150μg/mL HygromycinB) 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.