KC-1606

293T-ACEII Cell Line

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Background of 293T-ACEII Cell Line

ACEII is also known as ACEH, is an enzyme that belongs to the angiotensin-converting enzyme family of dipeptidyl carboxydipeptidases and has considerable homology to human angiotensin 1 converting enzyme. ACE2 is known to be expressed in various human organs, and its organ- and cell-specific expression suggests that it may play a role in the regulation of cardiovascular and renal function, as well as fertility. In addition, ACEII is a functional receptor for the spike glycoprotein of the human coronavirus HCoV-NL63 and the human severe acute respiratory syndrome coronaviruses, SARS-CoV and SARS-CoV-2, the latter is the causative agent of coronavirus disease-2019(COVID-19).

Specifications

Catalog Number	KC-1606
Cell Line Name	293T-ACEII Cell Line
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous ACEII 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 1:4-1:5 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-ACEII cell line was generated using a lentiviral vector expressing the ACEII sequence.

Characterization

Figure 1: Characterization of ACEII overexpression in the 293T-ACEII stable clone using qPCR.

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 1:4-1:5 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

"Gene: ACE2, angiotensin I converting enzyme 2". National Center for Biotechnology Information (NCBI). U.S. National Library of Medicine. 2020-02-28.
Hikmet F, Méar L, Edvinsson Å, Micke P, Uhlén M, Lindskog C (July 2020). "The protein expression profile of ACE2 in human tissues". Molecular Systems Biology. 16 (7): e9610. doi:10.15252/msb.20209610. PMC 7383091. PMID 32715618.
Hamming I, Timens W, Bulthuis ML, Lely AT, Navis G, van Goor H (June 2004). "Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis". The Journal of Pathology. 203 (2): 631–637. doi:10.1002/path.1570. PMC 7167720. PMID 15141377.
Kasmi Y, Khataby K, Souiri A (2019). "Coronaviridae: 100,000 Years of Emergence and Reemergence". In Ennaji MM (ed.). Emerging and Reemerging Viral Pathogens: Fundamental and Basic Virology Aspects of Human, Animal and Plant Pathogens. Vol. 1. Elsevier. p. 135. ISBN 978-0-12-819400-3.
Donoghue M, Hsieh F, Baronas E, Godbout K, Gosselin M, Stagliano N, et al. (September 2000). "A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9". Circulation Research. 87 (5): E1–E9.