AT1B Knockout Immortalized Mouse Renal Proximal Tubule Cell Line

The proximal tubule is part of the nephron duct system in the kidney, involved in regulating the renin-angiotension system. Cells of this region aid in regulating sodium and volume homeostasis as well as regulating the systemic blood pressure. The protein Angiotension II (Ang II) and its receptors AT₁ and AT₂ are some of the most studied proteins in this system. Even so, there is an incomplete understanding of the AT₁ receptors AT_1A and AT_1B. The AT1B Knockout Immortalized Mouse Renal Proximal Tubule Cell Line is a conditionally immortalized mouse renal proximal tubule cells isolated from the mouse harboring thermolabile mutation (tsA58) of the simian virus 40 large T antigen. The functional expression of the SV40 large T antigen is induced by culturing the cells in vitro in medium containing IFN γ at a temperature permissive (+33°C). At a non-permissive temperature (+37°C-+39°C), the cells cease to proliferate. When paired up with the wild type > (Cat. No. T0624) and other angiotensin receptor (Ang II)-deficient cell lines AT_1A ^-/- (Cat. No. T0626), AT_1A ^-/- AT_1B ^-/- (T0628) and AT₂ ^-/- (T0625) these cell lines represent valuable tools to study fluid and electrolyte transportation in the proximal tubule region.

Immortalization Method: C57B/6 mouse with AT_1B^-/- genotype

BioSafety Level: II

Organism: Mouse (M. musculus)

Species: Mouse

Source Organs: Kidney

Organ Type: Kidney

Growth Properties: Adherent

Morphology: Cobblestone

Seeding Density: Thaw entire contents into an appropriate T25 flask as specified in the Propagation instructions.

Propagation: The base medium for this cell line is Prigrow IV medium available at abm, Cat. No. TM004. To make the completed growth medium, add the following components to the base medium: fetal bovine serum (TM999) to a final concentration of 5%, 10 nM aldosterone, 50 µM L-ascorbic acid 2-phosphate, 4 µg/mL dexamethasone, 10 ng/mL epidermal growth factor, 5 µg/mL insulin, 20 nM sodium selenite (Na2SeO3), 5 µg/mL transferrin, 1 nM L-3,3’,5-triiodothyronine (T3), 10 U/mL recombinant mouse interferon gamma (IFN-γ) and Penicillin/Streptomycin Solution (G255) to a final concentration of 1%.

Carbon dioxide (CO₂): 5%, Temperature: +33.0°C.

Grow cells on 5% gelatine and 10 mg/mL laminin (diluted 1:1) coated vessels. To differentiate the cells, grow the cells at +37°C-+39°C in the absence of IFN-γ.

Quality Control: (1) RT-PCR was performed to confirm AT receptor genotype. (2) Electrical conductivity was measured to compare immortalized cells relative to non-immortalized counterparts. (3) Immunostaining was performed to determine localization of proteins associated with epithelial cells and assess morphology. (4) Changes in short circuit current were quantified to compare immortalized cell cotransporters to non-immortalized ones. (5) Western blotting was performed to detect the presence of AT receptor proteins. (6) Live cell microscopy of AngII-mediated β-arrestin 2 translocation was performed to measure receptor functionality.

Shipping Condition: Dry Ice

Storage Condition: liquid nitrogen or -180°C

Reference: Woost, P. G., Kolb, R. J., Finesilver, M., Mackraj, I., Imboden, H., Coffman, T. M., & Hopfer, U. (2006). Strategy for the development of a matched set of transport-competent, angiotensin receptor-deficient proximal tubule cell lines. in vitro cellular & developmental biology. Animal, 42(7), 189–200. https://doi.org/10.1290/0511076.1