KC-4554

293T-ACVR2A-ACVR2B-KO-rabbit-ACVR2B Cell Line

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Background of 293T-ACVR2A-ACVR2B-KO-rabbit-ACVR2B Cell Line

ACVR2B (Activin A Receptor Type 2B) is a Protein Coding gene. Activins are dimeric growth and differentiation factors which belong to the transforming growth factor-beta (TGF-beta) superfamily of structurally related signaling proteins. Activins signal through a heteromeric complex of receptor serine kinases which include at least two type I (I and IB) and two type II (II and IIB) receptors. These receptors are all transmembrane proteins, composed of a ligand-binding extracellular domain with cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine specificity. Type I receptors are essential for signaling; and type II receptors are required for binding ligands and for expression of type I receptors. Type I and II receptors form a stable complex after ligand binding, resulting in phosphorylation of type I receptors by type II receptors. Type II receptors are considered to be constitutively active kinases. This gene encodes activin A type IIB receptor, which displays a 3- to 4-fold higher affinity for the ligand than activin A type II receptor.

Specifications

Catalog Number	KC-4554
Cell Line Name	293T-ACVR2A-ACVR2B-KO-rabbit-ACVR2B Cell Line
NCBI/UniProt Accession Number	XM_051839742
Clone Number	7-5#
Host Cell Line	293T-ACVR2A-ACVR2B-KO
Description	293T-ACVR2A-ACVR2B-KO cell line stably expressing exogenous rabbit ACVR2B 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% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 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

Cell Line Generation

293T-ACVR2A-ACVR2B-KO-rabbit-ACVR2B cell line was generated using a lentiviral vector expressing the rabbit ACVR2B sequence.

Characterization

Figure 1: Characterization of rabbit ACVR2B overexpressing in 293T-ACVR2A-ACVR2B-KO stable clone using FACS.

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

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 1μg/mL Puromycin)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 1:4-1:5 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

Senanayake U, Das S, Vesely P, Alzoughbi W, Fröhlich LF, Chowdhury P, Leuschner I, Hoefler G, Guertl B. miR-192, miR-194, miR-215, miR-200c and miR-141 are downregulated and their common target ACVR2B is strongly expressed in renal childhood neoplasms. Carcinogenesis. 2012 May;33(5):1014-21. doi: 10.1093/carcin/bgs126. Epub 2012 Mar 19. PMID: 22431721.
O'Sullivan MP, Denihan N, Sikora K, Finder M, Ahearne C, Clarke G, Hallberg B, Boylan GB, Murray DM. Activin A and Acvr2b mRNA from Umbilical Cord Blood Are Not Reliable Markers of Mild or Moderate Neonatal Hypoxic-Ischemic Encephalopathy. Neuropediatrics. 2021 Aug;52(4):261-267. doi: 10.1055/s-0041-1725012. Epub 2021 Mar 11. PMID: 33706404.