KC-5496

3T3-L1-ALK7-High Cell Line

62544

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Background of 3T3-L1-ALK7-High 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-5496
Cell Line Name	3T3-L1-ALK7-High Cell Line
NCBI/UniProt Accession Number	NM_145259.3
Clone Number	1-68#
Host Cell Line	3T3-L1
Description	Stable 3T3-L1 cell line expressing exogenous human ALK7 gene in high 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% basal medium+20% FBS+10% DMSO
Propagation Medium	DMEM+10%FBS+1µg/mL puromycin+Glutamax 1X
Selection Marker	Puromycin
Morphology	Epithelial-like
Subculture	Split saturated culture 1:3-1:6 every 2-3 days
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

3T3-L1-ALK7-High cell line was generated using a lentiviral vector expressing the human ALK7 sequence.

Characterization

Figure 1: Characterization of ALK7 overexpression in the 3T3-L1-ALK7 stable clone using FACS.

Figure 2: Characterization of ALK7 in the 3T3-L1-ALK7 stable clone using PCR sequencing.

Cell Resuscitation

Pre-warm complete culture medium (basal medium and 10% FBS and 1µg/mL puromycin and Glutamax 1X) in a 37°C water bath.
Rapidly thaw the cryovial in a 37°C water bath for 1-2 minutes with gentle agitation.
Transfer the vial to a biosafety cabinet, and disinfect the exterior with 70% ethanol.
Aseptically transfer the cell suspension dropwise into a sterile centrifuge tube containing 9.0 mL of pre-warmed complete medium.
Centrifuge at approximately 125 × g for 5–7 minutes at room temperature, carefully aspirate the supernatant without disturbing the cell pellet.
Gently resuspend the pellet in an appropriate volume of complete medium and transfer the suspension into a T25 flask.
Incubate the flask in a 37°C in a humidified 5% CO₂ incubator.
Assess cell viability and morphology after 24 hours. If cells appear healthy, replace the medium with fresh medium supplemented with the appropriate selective antibiotic.
Subculture the cells at a ratio of 1:3-1:6 every 2-3 days upon reaching 80%–90% confluency.

Cell Freezing

Prepare the freezing medium (70% basal medium, 20% FBS and 10% DMSO) freshly before use.
Pre-chill the freezing medium on ice and label the cryovials accordingly.
Transfer the cell suspension to a sterile conical tube and perform a cell count to determine total viability and density.
Centrifuge the cells at 250×g for 5 minutes at room temperature; carefully aspirate the supernatant.
Gently resuspend the cell pellet in chilled freezing medium, ensuring a minimum cell density of 3×10⁶ cells/mL.
Aliquot 1 mL of the cell suspension into each pre-labeled cryovial.
Place the cryovials into a CoolCell® container and store at -80°C overnight for controlled-rate cooling.
Transfer the cryovials to the liquid nitrogen for long-term storage the following day.

References

1.Ibáñez CF. Regulation of metabolic homeostasis by the TGF-β superfamily receptor ALK7. FEBS J. 2022 Oct;289(19):5776-5797.
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.
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.
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.