Generating the putative Q₅₂, Q₃₁ and Q₁₀-GFP constructs using the Tol2-Q₈₀-GFP-v2A-GFP multicistronic reporter construct. a Vector map of the Tol2-Q₈₀-GFP-v2A-GFP construct. b Summary of CAA and CAG degenerate codon usage in this construct. c Chromatograph of the construct depicting the randomly interspaced glutamine coding CAG and CAA triplets. d Schematic illustration of PCR-based exclusion amplification of the Q₈₀-GFP containing construct to generate polyQ constructs with lower numbers of glutamine repeats. e Q₈₀ sequence showing Q₅₂, Q₃₁ and Q₁₀ primer binding sites. f Sequences of primers intended to generate Q₅₂, Q₃₁ and Q₁₀ constructs. g Analytical agarose gel electrophoresis for each of the putative Q₈₀, Q₅₂, Q₃₁ and Q₁₀ vectors using primers flanking the polyQ region. PCR product sizes of ~ 270, ~ 180, ~ 120 and ~ 60 bp for the intended putative Q₈₀, Q₅₂, Q₃₁ and Q₁₀-GFP constructs, respectively, were seen

Vector map and sequence analysis of the constructs coding for Q₅₂, Q₂₁ and Q₁₁-GFP generated by PCR based excision amplification. a Vector map of Tol2-Q₅₂-GFP-v2A-GFP construct. b Chromatograph of the Q₅₂ construct. c Vector map of Tol2-Q₂₁-GFP-v2A-GFP construct. d Chromatograph of the Q₂₁ construct. e Vector map of Tol2-Q₁₁-GFP-v2A-GFP construct. f Chromatograph of the Q₁₁ construct

Analysis of the Tol2-Q_X-GFP-v2A-GFP construct expression in D. rerio. Zebrafish embryos were injected with 25 ng/µL of the Tol2-Q_X-GFP-v2A-GFP construct and 25 ng/µL mRNA coding for Tol2 transposase. a Brightfield (top panels) and fluorescent (bottom panels) images of the left side view of zebrafish embryo head and trunk regions expressing Q₈₀, Q₅₂, Q₂₁ and Q₁₁-GFP, ~ 24 hpf. b Brightfield (top panels) and fluorescent (bottom panels) images of the left side view of zebrafish embryo trunk and tail regions expressing Q₈₀, Q₅₂, Q₂₁ and Q₁₁-GFP, ~ 24 hpf. c Western blot analysis of the expression of Q_x-GFP constructs in D. rerio. Proteins were isolated from ~ 24 hpf embryos expressing the Q₈₀, Q₅₂, Q₂₁ and Q₁₁-GFP constructs. The proteins were resolved on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was probed with an anti-GFP antibody. The “empty” Tol2 vector possesses an expressed GFP gene that is replaced during construct insertion. d Western blot quantification. The GFP intensity for each numbered band of the Western blot and the Q_x-GFP to free GFP ratio are presented