KC-5060

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

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

ACVR2A (Activin Receptor Type-2A) is a gene that encodes a key transmembrane receptor for the TGF-beta (Transforming Growth Factor-beta) superfamily.Enables several functions, including PDZ domain binding activity; activin binding activity; and transmembrane receptor protein serine/threonine kinase activity. Involved in BMP signaling pathway. Acts upstream of or within several processes, including Sertoli cell proliferation; copulation; and regionalization. Located in cell surface. Is active in plasma membrane. Is expressed in several structures, including central nervous system; early conceptus; gonad; gut; and sensory organ. Used to study Weissenbacher-Zweymuller syndrome. Human ortholog(s) of this gene implicated in Lynch syndrome and colon cancer. Orthologous to human ACVR2A (activin A receptor type 2A).

Specifications

Catalog Number	KC-5060
Cell Line Name	293T-ACVR2A-ACVR2B-KO-mouse-ACVR2A- Cell Line
NCBI/UniProt Accession Number	NM_007396.4
Clone Number	2#
Host Cell Line	293T
Description	Stable 293T-ACVR2A-ACVR2B-KO cell line expressing exogenous mouse-ACVR2A 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 1:4-1:8 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

Cell Line Generation

293T-ACVR2A-ACVR2B-KO-mouse-ACVR2A cell line was generated using lentivirus expressing mouse-ACVR2A sequence.

Characterization

Figure 1. Characterization of mouse-ACVR2A over-expression in the 293T-ACVR2A-ACVR2B-KO-mouse-ACVR2A stable clone using FACS.

Figure 2. Characterization of 293T-ACVR2A-ACVR2B-KO-mouse-ACVR2A 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 1:4-1:8 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

Ihn HJ, Kim DH, Oh SS, Moon C, Chung JW, Song H, Kim KD. Identification of Acvr2a as a Th17 cell-specific gene induced during Th17 differentiation. Biosci Biotechnol Biochem. 2011;75(11):2138-41. doi: 10.1271/bbb.110436. Epub 2011 Nov 7. PMID: 22056434.