Introduction of single nucleotide variations into the promoter region of the mouse kidney anion exchanger 1 gene downstream of a TATA box changes its transcriptional activity

Abstract

Abstract(Introduction) Distal renal tubular acidosis (dRTA) is characterized by an impairment of urine acidification and can be caused by variations in genes functioning in α-intercalated cells such as anion exchanger 1 (AE1). AE1 encodes the Cl-/HCO3-exchanger in erythrocytes (eAE1) and α-intercalated cells (kAE1). We previously reported that in human erythroid intron 3 containing the promoter region of human kAE1, a SNP (rs999716) 39 base pairs downstream of the TATA box showed a higher minor allele A frequency in incomplete dRTA patients and such a promoter region showed reduced activities, leading to the hypothesis that those with the A allele may express less kAE1, developing incomplete dRTA. Here, single nucleotide variations were introduced downstream of the TATA box in the murine erythroid intron 3 to investigate changes in the promoter activity for murine kAE1 mRNA. (Methods) The erythroid intron 3 of C57BL/6 was subcloned into the pGL4.17 reporter vector, leading to mu kAE1Pro-pGL4.17. Three types of G to A substitutions were introduced 33, 35, and 36 base pairs downstream of the TATA box in the murine kAE1 promoter region by inverse PCR (Var1, Var2, and Var3, respectively). The HEK 293 cells were transfected with vectors. After 24 hours, the firefly luciferase activity was determined. (Results and Discussion) The promoter activity of Var1, as well as that of Var2 to a lesser extent, was reduced compared with that of the wild-type. The introduction of variations such as Var1 and Var2 into the murine genome by genome editing may help to establish mouse models of incomplete dRTA.