KC-3849

293T-ACVR2B-KO-1A4 Cell Line

34900

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Background of 293T-ACVR2B-KO-1A4 Cell Line

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. Activin is also thought to have a paracrine or autocrine role in follicular development in the ovary. Within the receptor complex, the type-2 receptors act as a primary activin receptors (binds activin-A/INHBA, activin-B/INHBB as well as inhibin-A/INHA-INHBA). The type-1 receptors like ACVR1B act as downstream transducers of activin signals. Activin binds to type-2 receptor at the plasma membrane and activates its serine-threonine kinase. The activated receptor type-2 then phosphorylates and activates the type-1 receptor. Once activated, the type-1 receptor binds and phosphorylates the SMAD proteins SMAD2 and SMAD3, on serine residues of the C-terminal tail. Soon after their association with the activin receptor and subsequent phosphorylation, SMAD2 and SMAD3 are released into the cytoplasm where they interact with the common partner SMAD4. This SMAD complex translocates into the nucleus where it mediates activin-induced transcription. Inhibitory SMAD7, which is recruited to ACVR1B through FKBP1A, can prevent the association of SMAD2 and SMAD3 with the activin receptor complex, thereby blocking the activin signal. Activin signal transduction is also antagonized by the binding to the receptor of inhibin-B via the IGSF1 inhibin coreceptor.

Specifications

Catalog Number	KC-3849
Cell Line Name	293T-ACVR2B-KO-1A4 Cell Line
Host Cell Line	293T
Description	Stable 293T clone with human ACVR2B gene knockout, No.1A4
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% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS
Selection Marker	N/A
Morphology	Fibroblastoid cells growing as a monolayer
Subculture	Split the saturated culture at a ratio of 1:4~1:5 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-ACVR2B-KO-1A4 cell line was generated using CRISPR method.

Characterization

Figure 1: Characterization of 293T-ACVR2B-KO-1A4 Cell Line stable clone using FACS.

Figure 2: Characterization of 293T-ACVR2B-KO-1A4 Cell Line stable clone using PCR sequencing.

Figure 3: Characterization of 293T-ACVR2B-KO-1A4 cell Line stable clone using RT-PCR sequencing.

Cell Resuscitation

Prewarm the culture medium (DMEM supplemented with 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 to a 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 x g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the
Resuspend the cell pellet with the appropriate volume of complete medium and transfer the cell suspension
Incubate the flask in an incubator at 37°C, 5% CO₂.
Split the saturated culture at a ratio of 1:4-1:5 every 2-3 days; seed out at about 1-3 x10⁵ cells/mL.

Cell Freezing

Freshly prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) before use.
Keep the freezing medium on ice and label cryovials for later use.
Harvest cells in a sterile, conical centrifuge tube during the logarithmic growth period and count the cells.
Centrifuge the cells at room temperature at 250xg for 5 minutes and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3 x10⁶cells/ml in the 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

1. 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. 2. Attisano L, Wrana JL, Montalvo E, Massagué J. Activation of signalling by the activin receptor complex. Mol Cell Biol. 1996 Mar;16(3):1066-73. doi: 10.1128/MCB.16.3.1066. PMID: 8622651; PMCID: PMC231089.