Fig. 1: Self-excising reporters for ratiometric readout of exon-10-specific isoforms of human MAPT.
From: Non-invasive and high-throughput interrogation of exon-specific isoform expression
a, Scheme of the genomic organization of the human MAPT locus with the EXSISERS reporter NLuc inserted into the alternatively spliced exon 10 and FLuc inserted into the constitutive exon 11, both flanked with CCs and inteins, resulting in the ratiometric reporter system (EXSISERSMAPT:10NLuc-11FLuc), which detects the fractional expression of 4R referenced against total tau (pan-tau). b, Schematic of the post-translational splicing of 4R-tau; the intein-flanked NLuc and FLuc are spliced out post-translationally. NLuc specifically reports 4R-tau, and FLuc reports the presence of pan-tau. The excised luciferases can be read out independently through orthogonal substrates. c, Immunoblot analysis of dephosphorylated lysate from unmodified HEK293T cells and EXSISERSMAPT:10NLuc-11FLuc clonal cell lines containing either WT MAPT or the pathological IVS10+16 C>T mutation confirmed that both FLuc and NLuc were present only after MAPT induction. Anti-pan-tau antibodies revealed the pattern of all tau isoforms. The 4R-tau band was identified by excluding the bands that stained positive for 3R-tau. Data represent two independent experiments. MM, molecular mass. d, RLU of FLuc and NLuc luciferase was measured from both EXSISERSMAPT:10NLuc-11FLuc clones (WT and IVS10+16 C>T) without and with MAPT induction and normalized to the average RLU of the reference condition (WT with MAPT induction; striped bars, left y axis). The ratio of NLuc to FLuc (empty bars, right y axis) was then calculated for each sample (striped bars, left y axis). Data are mean ± s.d., n = 3 biological replicates. Selected results of Bonferroni MCT after two-way ANOVA are indicated by asterisks; ***P < 0.001, ****P < 0.0001 (full statistical results are provided in Supplementary Table 1). Source data are available online.
