Tol2-based On/Off/On (“Switchblade”, GBT-S1/S8) vectors for insertional mutagenesis and characterized gene trap lines.

<p><b>A.</b> Diagram of the vector and conditional regulation. The gene trap cassette is flanked by lox71, FRT-10, FRT+10 and lox66 sites. Components responsible for high degree of mutagenicity are shown in red. SA, carp beta actin splice acceptor, ^Gal-VP16, AUG-less Gal4-VP16, zp(A), zebrafish beta actin 3’ UTR and transcriptional termination sequences, cry, <i>X</i>. <i>laevis</i> gamma crystalline promoter, p(A), SV40 poly(A). The gene trap cassette is identical to that used in GBT-B1 gene trap vector (Balciuniene et al., 2013). Expression Flp recombinase will result in inversion of the gene trap cassette and one wild type (FRT) and one double mutant, inactive (FRT+10/-10, two red dots) site. Expression of the Cre recombinase will result in second inversion of the cassette conditionally mutating the gene. <b>B.</b> GBT-S1 and GBT-S8 gene trap lines. First column, gene trap line. Second column, sequence adjacent to the 3’ end of the gene trap integration. The capital CTG are the last three nucleotides of Tol2. Third column, location of the gene trap integration on the GRCz10 zebrafish genome assembly. Fourth column, insertionally mutated gene (IMG). Fifth column, sequence of the IMG-Gal4-VP16 fusion protein, IMG sequence is highlighted in aqua, Gal4 sequence is highlighted in magenta. The “linker” sequence is encoded by the linker between splice acceptor and Gal4 in GBT-S1 and GBT-S8. <b>C.</b> Diagram of gene trap loci. Gene trap integration site is shown as a triangle, with mutated gene’s exons depicted as squares: grey for non-coding, black for 5’ of integration site and white for 3’ of integration site. <b>D.</b> Assessment of transcript levels by quantitative RT-PCR. Three non-phenotypic lines were chosen for analysis. Quantitative RT PCR was performed on pools of RNA from wild type and homozygous mutant 5 dpf embryos, and normalized to beta actin. Error bars represent standard deviation.