Table 1.
Homozygous mouse models obtained by breeding mice with α1S and/or α2R mutations.
Table 2.
Primers and probes.
Fig 1.
Sequence alignment of murine ATP1A and ATP1A2 variants and recognition sequences of the PCR probes.
Amino acid numbering is based on current reference sequences for the murine NKA α1 and α2 subunits (from Table 1). ATP1A: gray highlight indicates the probe target site for the WT α1R mutant; red highlight indicates the probe target site for the sensitive α1S mutant. Base substitutions are indicated in red font. The presence or absence of vertical lines between sequences indicates whether a base change in the codon is conserved. ATP1A2: corresponding annotation. Blue highlight indicates the probe target site for the mutant α2R mutant. Green font indicates natural variance between C57BL/6J and 129S1/SImJ mouse strains.
Fig 2.
Detection of α1S and α2R mutants by qPCR using mutant-specific fluorescent probes.
ΔRn, fluorescence level vs. cycle number (CT). Horizontal lines indicate threshold fluorescence level for the WT (black), α1S (red) and α2R (blue) probes. Samples considered positive for a specific allele showed CT in the range of 26-31for the corresponding probe, while negative samples did not reach threshold in 35 cycles; heterozygous samples had amplification of both probes within 2 CT of each-other. The curves from both probes (run separately, in duplicate) of each gene are superimposed onto one plot. A) Amplification plots from the α1 (ATP1A1) assay of 3 samples representative of WT, HET, and homozygous mutant genotypes; α1R probe (black), α1S probe (red). B) Amplification plots from the α2 (ATP1A2) assay of 3 samples representative of WT, HET, and homozygous mutant genotypes; α2S probe (black), α2R probe (blue).