Fig. 1
Expanded G4C2 transgenes produce GFP-tagged GR. a. A new transgenic (G4C2)n model was developed to look at RAN-translation of the GR reading frame. A “leader” sequence (LDS) was added 5′ of the repeat: 114 bp of intronic sequence found upstream of the repeat in patient samples. The GR reading frame has an in-frame GFP coding sequence 3′ of the repeat that lacks an ATG initiation. b. To define the number of repeats inserted into genomes of w1118 transgenic flies, PCR reactions were developed that amplified the repeat and its flanking region. The number of repeats were calculated from PCR product lengths measured on a Bioanalyzer and agarose gel. Shown: the maximum number of repeats found in the control (CTRL) or expanded (EXP) G4C2 fly lines; individual data points from 2 independent DNA preps with mean. c. qPCR analysis for RNA levels between control and expanded G4C2 fly lines. Shown: individual data points with mean ± SD. Statistics: unpaired student t-test, p-value **** < 0.0001. d. Western immunoblots confirmed GR is produced and successfully tagged with GFP in EXP-G4C2 flies. No GR/GFP was detected in control G4C2 flies, even with overexposure (Additional file 1: Figure S1). Uncropped westerns (Additional file 1: Figure S4) e. External and internal eye analysis in animals expressing G4C2 or control (DSRED) transgenes using GMR-GAL4. Degeneration was seen only in LDS-(G4C2)EXP animals: external pigment loss and reduced integrity of internal retina tissue. f. External eye imaging for fluorescence caused by transgene expression. Positive (DSRED) control flies show uniform, diffuse signal. Control G4C2 flies show no signal, even with increased exposure (data not shown). Expanded G4C2 flies show GFP puncta. Shown (e-f): representative images while all conditions were tested 2+ times. For full genotypes see Additional file 7: Table S1