Fig. 1: Imaging Dmd and Utrn expression in vivo using bioluminescence.

a, b Schematic of the knock in (KI) strategy used to generate Dmd^G (a) and Utrn^R (b) reporter mouse lines. Reporter genes were inserted into the final exon of the endogenous genes, between the stop codon and the 3’UTR. a The CBG99Luc gene was knocked into endogenous Dmd, separated from the coding region of exon 79 with a T2A site. b LacZ and RFluc genes were knocked into endogenous Utrn, separated by T2A sites. c Bioluminescent imaging of adult (P28) heterozygous Dmd^G+/− female mice showed strong bioluminescent signal corresponding with the location of the musculature and head, while no signal was visible in a wild type (WT) control mouse (Dmd^G−/−). d Quantification of whole-body radiance emitted from Dmd^G+/+, Dmd^G+/−, and WT mice showed that bioluminescence emitted from homozygous (Dmd^G+/+) females was around double that emitted by hemizygous (Dmd^G+/−) females and equivalent to Dmd^G+/y males. Significantly more bioluminescence was detected in all Dmd^G+ mice than in WT animals. Bar graphs show mean + /− SEM. For each sex, comparisons between genotypes were performed using one-way ANOVA (p < 0.0001) with Sidak’s multiple comparisons test (adjusted p values: **p < 0.01, ***p < 0.001, ****p < 0.0001). e Ex vivo bioluminescent imaging of tissues dissected from heterozygous (Dmd^G+/−) female mice; highest levels of radiance (blue green) were observed in the skeletal muscle (SkM) and heart, with lower signal in white adipose (WAT) and kidney. Quantification (below) shows mean radiance (p/s/cm²/sr) (n = 3 + /− SD). f Allele-specific RT-qPCR, using primers that distinguish mRNA derived from the KI allele (Dmd^G+, white bars) and the WT allele (Dmd^G−, grey bars); comparable expression of both alleles was observed in tissues from adult Dmd^G+/− female mice. g Bioluminescence in adult (P28) heterozygous (Utrn^R+/−) female mice was detected particularly across the abdomen and head, but not in the WT (UtrnR^−/−) animal. h Quantification of whole-body radiance emitted from Utrn^R+/+, Utrn^R+/− and WT mice. For both sexes, homozygous (Utrn^R+/+) mice emitted approximately twice the flux of heterozygous (Utrn^R+/−) mice. Radiance was significantly higher in all Utrn^R+ mice than WT mice (Utrn^R−/−). Statistical analysis performed as of d. i Bioluminescence imaging of ex vivo tissues from Utrn^R+/− mice showed strong signal in WAT and kidney, but less in the SkM and heart. Quantification (below) shows mean radiance (p/s/cm²/sr) (n = 3 + /− SD). j Allele-specific RT-qPCR on tissues dissected from heterozygous (Utrn^R+/−) mice showed comparable mRNA expression from the targeted allele (Utrn^R+, white bars) and the wildtype allele (Utrn^R−, grey bars). k Expression of lacZ in tissues from Utrn^R+/− mice was quantified by RT-qPCR. f, j, k Expression levels were normalised to 18 S and Tbp and are shown relative to the skeletal muscle sample. Bar graphs show mean + /− SEM.