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. 2019 Dec 13;9:19045. doi: 10.1038/s41598-019-55659-w

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Experimental scheme testing the integration of the gfp gene into the Stenostomum genome. (A) SL0-sm01 electroporated worms using pBac [3xp3-EGFPafm] and pB∆Sac plasmids were tested by PCR (gel lanes: L = Kb ladder; N = control/ no DNA; 1 = worm sample three days after electroporation; 2 = worm sample 15 days after electroporation). Amplicons of 2,700 bp indicated that precise transposition had not occurred (see scheme C). Illumina NSG using gDNA isolated six months after electroporation allowed assembly of both plasmids (complete and circularized). However, no indication of integration into the worm’s chromosomes was obtained. (B) SL0-sm02 electroporated worms using pBac[3xp3-EGFPafm] and pB∆Sac plasmids tested by PCR 3 and 15 days after electroporation. No amplicons were obtained 15 days after electroporation, showing the plasmid was lost in this period; (C) scheme of the pBac[3xp3-EGFPafm] plasmid containing piggyBac TIRs sequences (red arrowhead) and gfp gene (green), other plasmid sequences (blue) and primers used for PCRs (orange arrows). Amplicons containing 81 bp of plasmid sequences upstream of the TIRs, suggest that a precise transposition did not occur.