KC-4427

293T-ASGR1-Cell-Line

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Background of 293T-ASGR1-Cell-Line

Asialoglycoprotein receptor 1 (ASGR1) is a major subunit of ASGPR (also known as Ashwell-Morell receptor, Ashwell receptor or hepatic lectin) and is highly conserved among mammals; it is a Protein Coding gene. It is a transmembrane protein specifically expressed in hepatocytes and plays a pivotal role in maintaining circulating glycoprotein homeostasis. It plays a critical role in serum glycoprotein homeostasis by mediating the endocytosis and lysosomal degradation of glycoproteins with exposed terminal galactose or N-acetylgalactosamine residues. Studies have suggested that asialoglycoprotein receptor 1 (ASGR1) increased cholesterol efflux and thus lowered blood cholesterol and reduced atherosclerosis. Diseases associated with ASGR1 include Hepatitis and Hepatitis B.

Specifications

Catalog Number	KC-4427
Cell Line Name	293T-ASGR1-Cell-Line
Host Cell Line	293T
Description	Stable HEK293T clone expressing exogenous human ASGR1 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
In Vivo Validation	NA

Cell Line Generation

293T-ASGR1 cell line was generated using a lentiviral vector expressing the human ASGR1 sequence.

Characterization

Figure 1: Characterization of human ASGR1 overexpression in the 293T ASGR1 stable clone using FACS.

Figure 2: Characterization of human ASGR1 in the 293T ASGR1 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

Zhang, Z., Leng, X.K., Zhai, Y.Y. et al. Deficiency of ASGR1 promotes liver injury by increasing GP73-mediated hepatic endoplasmic reticulum stress. Nat Commun 15, 1908 (2024).
Zhao, W., Xu, S. & Weng, J. ASGR1: an emerging therapeutic target in hypercholesterolemia. Sig Transduct Target Ther 8, 43 (2023).
Wang JQ, Li LL, Hu A, Deng G, Wei J, Li YF, Liu YB, Lu XY, Qiu ZP, Shi XJ, Zhao X, Luo J, Song BL. Inhibition of ASGR1 decreases lipid levels by promoting cholesterol excretion. Nature. 2022 Aug;608(7922):413-420. doi: 10.1038/s41586-022-05006-3. Epub 2022 Aug 3. PMID: 35922515.