KC-3733

293T-AQP4-M1 Cell Line

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Background of 293T-AQP4-M1 Cell Line

This gene encodes a member of the aquaporin family of intrinsic membrane proteins that function as water-selective channels in the plasma membranes of many cells. This protein is the predominant aquaporin found in brain and has an important role in brain water homeostasis. Alternatively spliced transcript variants encoding different isoforms have been described for this gene. Additional isoforms, resulting from the use of alternative in-frame translation initiation codons, have also been described. Recent studies provided evidence for translational readthrough in this gene, and expression of C-terminally extended isoforms via the use of an alternative in-frame translation termination codon.

Specifications

Catalog Number	KC-3733
Cell Line Name	293T-AQP4-M1 Cell Line
NCBI/UniProt Accession Number	NM_001650.7
Clone Number	1#
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous AQP4-M1 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-AQP4-M1 cell line was generated using a lentiviral vector expressing the AQP4-M1 sequence.

Characterization

Figure 1: Characterization of AQP4-M1 overexpression in the 293T-AQP4-M1 stable clone using qPCR.

Figure 2: Characterization of AQP4-M1 overexpressing in 293T-AQP4-M1 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 1:4-1:5 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

Dardiotis E, Paterakis K, Tsivgoulis G, Tsintou M, Hadjigeorgiou GF, Dardioti M, Grigoriadis S, Simeonidou C, Komnos A, Kapsalaki E, Fountas K, Hadjigeorgiou GM. AQP4 tag single nucleotide polymorphisms in patients with traumatic brain injury. J Neurotrauma. 2014 Dec 1;31(23):1920-6. doi: 10.1089/neu.2014.3347. Epub 2014 Oct 10. PMID: 24999750; PMCID: PMC4238262.
Owens GP, Ritchie A, Rossi A, Schaller K, Wemlinger S, Schumann H, Shearer A, Verkman AS, Bennett JL. Mutagenesis of the aquaporin 4 extracellular domains defines restricted binding patterns of pathogenic neuromyelitis optica IgG. J Biol Chem. 2015 May 8;290(19):12123-34. doi: 10.1074/jbc.M115.647149. Epub 2015 Mar 19. PMID: 25792738; PMCID: PMC4424347.