KC-5073

CHOK1-ALK7-Middle Cell Line

62267

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Background of CHOK1-ALK7-Middle Cell Line

ACVR1C (Activin A Receptor Type 1C) also known as ALK7, ALK7 (Activin receptor-like kinase 7) is a member of the TGF-β receptor superfamily predominantly expressed by cells and tissues involved in endocrine functions, such as neurons of the hypothalamus and pituitary, pancreatic β-cells and adipocytes. The TGFß superfamily receptor ALK7 is a suppressor of tumorigenesis and metastasis, Activation in neoplastic cells of the ALK7 signaling pathway by its principal ligand activin B induces apoptosis. Diseases associated with ACVR1C include Extraocular Retinoblastoma and Acquired Hyperkeratosis.

Specifications

Catalog Number	KC-5073
Cell Line Name	CHOK1-ALK7-Middle Cell Line
NCBI/UniProt Accession Number	NM_145259.3
Clone Number	32#
Host Cell Line	CHOK1
Description	Stable CHOK1 clone expressing exogenous human ALK7 gene in middle level
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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640+ 10% FBS + 10μg/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 24 hours
Mycoplasma Status	Negative

Cell Line Generation

CHOK1-ALK7-Middle cell line was generated using a lentiviral vector expressing the human ALK7 sequence.

Characterization

Figure 1: Characterization of human ALK7 overexpression in CHOK1-ALK7-Middle stable clones using FACS.

Figure 2: Characterization of CHOK1-ALK7-Middle cell line stable clone using PCR sequencing

Cell Resuscitation

1. Prewarm culture medium (RPMI1640+ 10% FBS + 10μg/mL Puromycin)in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO₂ incubator.
8. Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage.

References

1. Ibáñez CF. Regulation of metabolic homeostasis by the TGF-β superfamily receptor ALK7. FEBS J. 2022 Oct;289(19):5776-5797. doi: 10.1111/febs.16090. Epub 2021 Jul 11. PMID: 34173336.
2. Gong FH, Cheng WL, Zhang Q, Chen XL, Cao JL, Yang T, Song WH, Zhao F. ALK7 Promotes Vascular Smooth Muscle Cells Phenotypic Modulation by Negative Regulating PPARγ Expression. J Cardiovasc Pharmacol. 2020 Aug;76(2):237-245. doi: 10.1097/FJC.0000000000000857. PMID: 32467530.
3. Michael IP, Saghafinia S, Tichet M, Zangger N, Marinoni I, Perren A, Hanahan D. ALK7 Signaling Manifests a Homeostatic Tissue Barrier That Is Abrogated during Tumorigenesis and Metastasis. Dev Cell. 2019 May 6;49(3):409-424.e6. doi: 10.1016/j.devcel.2019.04.015. PMID: 31063757.
4. Goebel EJ, Ongaro L, Kappes EC, Vestal K, Belcheva E, Castonguay R, Kumar R, Bernard DJ, Thompson TB. The orphan ligand, activin C, signals through activin receptor-like kinase 7. Elife. 2022 Jun 23;11:e78197. doi: 10.7554/eLife.78197. PMID: 35736809; PMCID: PMC9224996.