KC-5415

293T-INSRA Cell Line

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Background of 293T-INSRA Cell Line

INSRA actually refers to "Insulin Receptor A" (Insulin receptor a), which is a subtype or nomenclature of the insulin receptor gene (INSR) in specific species.INSR is a Protein Coding gene.This gene encodes a member of the receptor tyrosine kinase family of proteins. The encoded preproprotein is proteolytically processed to generate alpha and beta subunits that form a heterotetrameric receptor. Binding of insulin or other ligands to this receptor activates the insulin signaling pathway, which regulates glucose uptake and release, as well as the synthesis and storage of carbohydrates, lipids and protein. Mutations in this gene underlie the inherited severe insulin resistance syndromes including type A insulin resistance syndrome, Donohue syndrome and Rabson-Mendenhall syndrome. Alternative splicing results in multiple transcript variants.

Specifications

Catalog Number	KC-5415
Cell Line Name	293T-INSRA Cell Line
NCBI/UniProt Accession Number	NM_001079817.3
Clone Number	1-8#
Host Cell Line	293T
Description	293T cell line stably expressing exogenous INSRA 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 + 1 μg/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-INSRA cell line was generated using a lentiviral vector expressing the INSRA sequence.

Characterization

Figure1: Characterization of INSRA overexpressing in 293T stable clones using FACS.

Figure2: Characterization of 293T-INSRA cell line stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS + 1 μg/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

Westermeier F, Salomón C, Farías M, Arroyo P, Fuenzalida B, Sáez T, Salsoso R, Sanhueza C, Guzmán-Gutiérrez E, Pardo F, Leiva A, Sobrevia L. Insulin requires normal expression and signaling of insulin receptor A to reverse gestational diabetes-reduced adenosine transport in human umbilical vein endothelium. FASEB J. 2015 Jan;29(1):37-49. doi: 10.1096/fj.14-254219. Epub 2014 Oct 28. PMID: 25351985.
Yuasa T, Amo K, Ishikura S, Nagaya H, Uchiyama K, Hashida S, Ebina Y. Development of in vitro model of insulin receptor cleavage induced by high glucose in HepG2 cells. Biochem Biophys Res Commun. 2014 Feb 28;445(1):236-43. doi: 10.1016/j.bbrc.2014.01.187. Epub 2014 Feb 6. PMID: 24508798.
Osawa H, Nishimiya T, Ochi M, Niiya T, Onuma H, Kitamuro F, Kaino Y, Kida K, Makino H. Identification of novel C253Y missense and Y864X nonsense mutations in the insulin receptor gene in type A insulin-resistant patients. Clin Genet. 2001 Mar;59(3):194-7. doi: 10.1034/j.1399-0004.2001.590309.x. PMID: 11260230.