Polymerase chain reaction (PCR) -based detection of excised Tol1 and Tol2 donor plasmids. Each gel panel contains PCR products amplified from six embryos microinjected with a donor plasmid and transposase mRNA. The leftmost lane consists of DNA fragments from the GeneRules^TM 50-bp DNA ladder (Fermentas). Top row, PCR assay to detect 283-bp or 321-bp fragments from excised pDon[Tol1] and pDon[Tol2] plasmids, respectively. Middle row, PCR assay to detect 316-bp and 546-bp fragments from unexcised pDon[Tol1] and pDon[Tol2] constructs, respectively. Bottom row, PCR amplification of a 428-bp fragment of the β-lactamase gene from the vector backbone. Black arrowheads indicate sizes of expected fragments.

Tol1-mediated germline transgenesis in zebrafish. Green fluorescent protein (GFP) expression in transgenic G1 embryos and larvae at various developmental stages is consistent with germline transmission of one or more chromosomally integrated Tol1-GFP expression cassettes.

Southern blot hybridization analysis of Tol1 insertions in G1 fish. G01/G1/1 to G01/G1/4 and G02/G1/1 to G02/G1/4 were green fluorescent protein (GFP) -positive G1 fish from founder fish G01 and G02, respectively. Genomic DNAs were extracted from whole fish. Black arrowheads indicate the hybridization bands detected in each lane. The leftmost lane contains 10 pg of pDon126 DNA digested with Sal1, which releases a ∼2.9-kb fragment containing the Tol1 left arm. Genomic DNA from a GFP-positive G0 fish (lane 2), uninjected zebrafish (lane 3) and medaka (lane 12) were also included in the analysis. Between one and three copies of Tol1 insertions were detected in G01/G1/1 to G01/G1/4 fish, while one to four copies were found in G02/G1/1 to G02/G1/4 fish.