Location of <i>piggyBac</i> integration sites in AegPB8.

*<p>Position of underlined nucleotide shown based on <i>Aedes aegypti</i> genome version 66.1 (AegL1)</p

Plasmids used in this study.

<p>All plasmids are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068454#s2" target="_blank">Material and Methods</a>. Large arrowheads represent the terminal sequences of <i>piggyBac</i>. Act5C, promoter from <i>D. melanogaster</i> gene <i>Actin5C; Hygromycin^R,</i> coding region for bacterial gene <i>hygromycin B phosphotransferase</i>; ie1, promoter from the baculovirus gene <i>immediate early 1</i>; Amp^R, bacterial gene <i>beta-lactamase</i>; hsp70, promoter from <i>D. melanogaster</i> gene <i>hsp70</i>; PB-transposase, coding region for <i>piggyBac</i> transposase; DsRed, coding region for <i>Discosoma sp</i>. gene <i>red fluorescent protein</i>; pUb, promoter from <i>D. melanogaster</i> gene <i>pUbi-p63e</i>; Kan^R, bacterial gene <i>Neomycin phosphotransferase II</i>; gDNA, refers to <i>Aedes aegypti</i> genomic DNA flanking the 5′ and 3 ends of <i>piggyBac</i> elements integrated in the genome of cell line AagPB8 (in pCL1w+) and in transgenic line 40D (in p40Dw+; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068454#pone.0068454-Sethuraman1" target="_blank">[25]</a>); mini-white, the <i>D. melanogaster</i> gene <i>w^+mW.hs;</i> attB, the bacterial attachment site for phage <i>ΦC31.</i></p

Plasmid-based <i>piggyBac</i> excision assay.

<p>A) Diagrammatic representation of the <i>piggyBac</i>-containing plasmid, <i>piggyBac</i> 3×P3EGFP used in the excision assay described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068454#s2" target="_blank">Material and Methods</a> (donor plasmid), and the same plasmid following precise excision of the <i>piggyBac</i> element (excision plasmid). The <i>piggyBac</i>-containing donor plasmid, <i>piggyBac</i> 3×P3EGFP, and <i>piggyBac</i> transposase expressing helper plasmid, pHspPBtpase:PubDsRed, were co-transfected into AagPB8 cells. Transfected cells were heat-shocked after 12 hrs and collected after 72 hrs. DNA was extracted and used as a template for PCR. Primers 1 and 2 (shown as labeled short half-arrows) were specific to the donor-plasmid backbone (494donorFWD, 494donorREV) and yield a 751 bp product (grey line) in the presence of the donor and excision plasmids. Primers 3 and 4 (494excisionFWD, 494excisionREV and shown as labeled short half-arrows) are specific to the plasmid DNA flanking the <i>piggyBac</i> element, however under the conditions of this experiment PCR products were only detected if donor plasmids missing the <i>piggyBac</i> element through excision were present, yielding a 540 bp PCR product (grey line). The 5′ and 3′ terminal <i>piggyBac</i> sequences are represented by arrows (5′PB, 3′PB). The duplicated TTAA target sequence into which <i>piggyBac</i> integrated is shown as a black diamond and the 3×P3EGFP transgene within the <i>piggyBac</i> element is shown as a black rectangle. The normally circular plasmids are represented as linear molecules. B) The PCR results from two <i>piggyBac</i> excision assays in AagPB8 cells. Lanes 1 and 2: from cells transfected with donor and <i>pHspPBtpase:PubDsRed</i> (2 independent transfections). Lane 3: from cells transfected with donor and control plasmids (pBluescript SKII+). Lane 4 and 5: positive controls for detecting excision events. The DNA used as a template in these reactions was a purified excision plasmid recovered from a previous excision assay (2 independent transfections). Lane 6: negative control for detecting excision events. DNA used as a template in this reaction came from cells transfected with donor plasmid only, without the transposase helper plasmid. Two PCR reactions were performed on each sample using primer combinations indicated above the lanes numbers. Primers 1+2 (same primers referred to in panel A) detected the presence of donor and excision plasmids and yielded a 751 bp reaction product (white arrow). Primers 3+4 (same primers referred to in panel A) yielded a 540 bp reaction product (white arrow) only when the <i>piggyBac</i> element in the donor plasmid had excised. Only the 540 and 751 bp bands are specific reaction products.</p

<i>piggyBac</i> transposable element display results using DNA isolated from cell line AagPB8.

<p>Lanes 1 are the results using DNA as a template isolated from cell line AagPB8 and shows evidence of the 5′ end of one of the two <i>piggyBac</i> elements that had integrated by canonical cut-and-paste transposition –80 bp band. The 5′ end of the second <i>piggyBac</i> element that integrated by canonical cut-and-paste transposition is not visible. This element can be detected when the 3′ ends of integrated <i>piggyBac</i> elements are visualized using transposable element display (not shown). The band at 250 bp is the <i>piggyBac</i> element associated with a copy of the integrated plasmid pBac:Act5cHyg:ie1EGFP. The sample was loaded into two adjacent lanes. Lanes 2 are the results using DNA as a template isolated from non-transgenic Aag-2 cells and this serves as a negative control for this assay since there are no <i>piggyBac</i> elements in <i>Ae. aegypti</i>. The sample was loaded into two adjacent lanes. Lanes 3 are the results using DNA as a template from AagPB8 cells 72 hours after being transfected with <i>piggyBac</i>-transposase-expressing pHspPBtpase:PubDsRed. The sample was loaded into two adjacent lanes. There was no evidence of <i>piggyBac</i> elements in other positions in the genome in Lane 3 as would be expected if <i>piggyBac</i> transposase mobilized the integrated <i>piggyBac</i> elements in AagPB8 cells. The asterisk indicated the position of a non-specific TE display band present in all samples. The positions of molecular weight markers 80 bp and 250 bp in length are shown.</p