KC-4548

293T-SMAD-Luc2-ACVR2A-ACVR2B-KO-rat-ACVR2A-ACVR2B Cell Line

60575

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Background of 293T-SMAD-Luc2-ACVR2A-ACVR2B-KO-rat-ACVR2A-ACVR2B Cell Line

This gene encodes a receptor that mediates the functions of activins, which are members of the transforming growth factor-beta (TGF-beta) superfamily involved in diverse biological processes. 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. The encoded type II receptor is primarily involved in ligand-binding and includes an extracellular ligand-binding domain, a transmembrane domain and a cytoplasmic serine-threonine kinase domain. This gene may be associated with susceptibility to preeclampsia, a pregnancy-related disease which can result in maternal and fetal morbidity and mortality.Diseases associated with ACVR2A include 2Q23.1 Microduplication Syndrome and Fibrodysplasia Ossificans Progressiva. Among its related pathways are Signaling by TGFβ family members and Signaling by NODAL. Gene Ontology (GO) annotations related to this gene include transferase activity, transferring phosphorus-containing groups and protein tyrosine kinase activity. An important paralog of this gene is ACVR2B.Diseases associated with ACVR2B include Heterotaxy, Visceral, 4, Autosomal and Visceral Heterotaxy. Among its related pathways are Akt Signaling and Signaling by TGFB family members. Gene Ontology (GO) annotations related to this gene include transferase activity, transferring phosphorus-containing groups and protein tyrosine kinase activity.

Specifications

Catalog Number	KC-4548
Cell Line Name	293T-SMAD-Luc2-ACVR2A-ACVR2B-KO-rat-ACVR2A-ACVR2B Cell Line
Clone Number	1C3
Host Cell Line	293T-SMAD-Luc2-ACVR2A-ACVR2B-KO
Description	Stable 293T-SMAD-Luc2-ACVR2A-ACVR2B-KO cell line expressing exogenous luciferase under the control of rat-ACVR2A-ACVR2B signaling pathway.
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 + 150μg/mL Hygromycin B + 1μg/mL Puromycin
Selection Marker	Hygromycin B, Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1 : 10 every 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 30 hours
Mycoplasma Status	Negative

Cell Line Generation

293T-SMAD-Luc2-ACVR2A-ACVR2B-KO-rat-ACVR2A-ACVR2B Cell Line was generated using a lentiviral vector expressing the rat ACVR2A-ACVR2B sequence.

Characterization

Figure1: Characterization of rat-ACVR2A-ACVR2B overexpression in 293T-SMAD-Luc2-ACVR2A-ACVR2B-KO cell line stable clones using FACS.

Figure2: Characterization of rat-ACVR2A-ACVR2B overexpression in 293T-SMAD-Luc2-ACVR2A-ACVR2B-KO cell line stable clones using RT-PCR sequencing.

Figure3: 293T-SMAD-Luc2-ACVR2A-ACVR2B-KO-rat-ACVR2A-ACVR2B cells were seeded into 96-well plates, treated with Activin A for 16 hours, and then read out using Bright-Glo Detection System.

Figure4: 293T-SMAD-Luc2-ACVR2A-ACVR2B-KO-rat-ACVR2A-ACVR2B cells were seeded into 96-well plates, treated with Bimagrumab(Cat#KB-1390, Kyinno) for 1 hours, and then treated with Activin A in the concentration of 50ng/mL for 16 hours, and then read out using Bright-Glo Detection System.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS, 150µg/mL Hygromycin B + 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 : 10 every 3 days; seed out at about 1 × 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

D'Abronzo FH, Swearingen B, Klibanski A, Alexander JM. Mutational analysis of activin/transforming growth factor-beta type I and type II receptor kinases in human pituitary tumors. J Clin Endocrinol Metab. 1999 May;84(5):1716-21. doi: 10.1210/jcem.84.5.5704. PMID: 10323406.
Zeybek B, Celik HA, Aydin HH, Askar N. Polymorphisms in the activin A receptor type 2A gene affect the onset time and severity of preeclampsia in the Turkish population. J Perinat Med. 2013 Jul;41(4):389-99. doi: 10.1515/jpm-2012-0187. PMID: 23633461.
Zhang H, Ruan Q, Chen C, Yu H, Guan S, Hu D, Yang C, Lin R, Zhuo C. Activin A/ACVR2A axis inhibits epithelial-to-mesenchymal transition in colon cancer by activating SMAD2. Mol Carcinog. 2023 Oct;62(10):1585-1598. doi: 10.1002/mc.23601. Epub 2023 Jun 28. PMID: 37378449.
Ishikawa S, Kai M, Murata Y, Tamari M, Daigo Y, Murano T, Ogawa M, Nakamura Y. Genomic organization and mapping of the human activin receptor type IIB (hActR-IIB) gene. J Hum Genet. 1998;43(2):132-4. doi: 10.1007/s100380050054. PMID: 9621519.