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. 2013 Oct 25;8(10):e77647. doi: 10.1371/journal.pone.0077647

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© 2013 Juntti et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Transgenic 4(B), while a wild-type sibling does not (A). Brightfield images of the larvae depicted in A and B are provided for comparison (C, D). E) Splinkerette nested PCR amplifies region surrounding transgene insertion site with primers in the splinkerette adapter (Splink3, Splink6), combined with primers at either the 5′ (Tol2-r, Splink7) or the 3′ (Splink5, Splink8) end of the transgenic construct. All bands were sequenced; arrow indicates band that provided sufficient quality sequence for mapping. F) Schematic of locations for primers in the Nkcrg2 locus and the integrated EF1α:GFP cassette. G) PCR from genomic DNA template of line 426 GFP+ fish and wild-type siblings demonstrating EF1α:GFP integration at the Nkcrg2 locus. H) Sequencing of PCR products shows that the EF1α:GFP cassette integrated on scaffold 3423. Sequence in red matches the Tol2 flanking region from pT2KXIGin, while sequence in black matches the A. burtoni genome. The boxed sequence is an 8 bp duplication of the genomic locus indicative of Tol2-catalyzed integration.

Inline graphic — Transgenic 4(B), while a wild-type sibling does not (A). Brightfield images of the larvae depicted in A and B are provided for comparison (C, D). E) Splinkerette nested PCR amplifies region surrounding transgene insertion site with primers in the splinkerette adapter (Splink3, Splink6), combined with primers at either the 5′ (Tol2-r, Splink7) or the 3′ (Splink5, Splink8) end of the transgenic construct. All bands were sequenced; arrow indicates band that provided sufficient quality sequence for mapping. F) Schematic of locations for primers in the Nkcrg2 locus and the integrated EF1α:GFP cassette. G) PCR from genomic DNA template of line 426 GFP+ fish and wild-type siblings demonstrating EF1α:GFP integration at the Nkcrg2 locus. H) Sequencing of PCR products shows that the EF1α:GFP cassette integrated on scaffold 3423. Sequence in red matches the Tol2 flanking region from pT2KXIGin, while sequence in black matches the A. burtoni genome. The boxed sequence is an 8 bp duplication of the genomic locus indicative of Tol2-catalyzed integration.