Generation of Mice Expressing RFP‐tagged Sodium Phosphate Cotransporter NaPi‐IIa

Abstract

Homeostasis of inorganic phosphate (Pi) is essential for various physiologic functions. The sodium phosphate cotransporter NaPi‐IIa is located in the brush border membrane (BBM) of the renal proximal tubule and represents the major renal Pi transporter. The plasma concentration of Pi is determined by factors regulating NaPi‐IIa abundance at the BBM. We are developing a novel mouse model expressing NaPi‐IIa tagged with red fluorescent protein (RFP) to analyze the regulation of the cotransporter at the BBM in real time and space and at the molecular level. We have generated RFP‐NaPi‐IIa constructs containing the RFP at three different locations of the cotransporter, the very N‐terminus, within the N‐terminal tail and within the big extracellular loop. Control experiments in opossum kidney cells and in Xenopus laevis oocytes to analyze the cotransporter polarization, PTH sensitivity, Na+‐dependent cotransport activity and kinetic characterization revealed that the RFP within the N‐terminal tail did not impair any of the analyzed parameters. Upon identification of the proper insertion site, we generated three pairs of transcription activator‐like effector nucleases (TALENs) predicted to cut the Slc34a1 (NaPi‐IIa) gene within the desired sequence. The pair with the highest cutting efficiency was chosen for the generation of RFP‐NaPi‐IIa mice. Consequently, we produced RFP donor DNA containing 5' and 3' homologous arms, as well as the RFP coding sequence. TALENs and the RFP donor were injected into one‐cell stage embryos to generate transgenic mice.This work was supported by NCCR Kidney.CH