Figure 1

PCR-based assays for transposon excision in T. dalmanni embryos. (A) Schematic representation of a PCR-based excision assay. Donor plasmids contain the terminal inverted repeats (TIRs), from the piggyBac, mariner or Minos transposon, flanking a transgene. In the presence of transposase, donor plasmids undergo excision of the transgene. "Excision primers" (red arrows) flank the entire construct including the TIRs. PCR of the unexcised construct will give rise to a product too large to be amplified efficiently (2-10 kb) under standard conditions. Amplification post-excision produces a smaller product (0.1-1 kb). Excision primers are validated using modified donor plasmids, from which the element had been excised by digestion with an appropriate restriction enzyme, as control templates. "Extraction primers" (blue arrows) amplify part of the donor plasmid backbone to demonstrate successful extraction of the donor plasmid from injected embryos. PCR with these primers produces the same size product (0.1-1 kb) both pre-and post-excision. (B-E) Results of the excision assay using piggyBac (B), mariner (C) and Minos (D and E). Templates for PCR were: DNA extracted from embryos injected with donor plasmid and a source of transposase (with transposase); DNA extracted from embryos with donor plasmid without a source of transposase (without transposase); donor plasmid control templates (+ve control); water (-ve control). PCR reactions either used excision primers (red lettering) to test for excision of the transposable element, or extraction primers (blue lettering) to test for successful extraction of the plasmids from injected embryos (see Additional file 2: Table S1). White triangles denote the expected size of the excision primer PCR product post-excision of the element. For the piggyBac (B) and mariner (C) assays, a DNA source of transposase was used. For the Minos assays both a DNA source of transposase (D) and an mRNA source of transposase were tested (E). Excision was detected for piggyBac and both Minos assays when the donor plasmid was injected with a source of transposase but not in the mariner assay. No excision was detected when donor plasmids were injected without a source of transposase indicating a lack of endogenous transposase activity in the embryos. In all cases donor plasmids were successfully extracted from embryos.