Expression of the zygotic gene, isg15, in zebrafish embryos after invasion by the plasmid, pEGFP-N1. A Heatmap to show normalized expression count differences of immune related genes from transcriptome sequencing at 6 hpf after injection of the foreign plasmid, pEGFP-N1, into 1-cell stage embryos. B Expression levels of six genes related to the apoptosis pathway, endogenous immune response and tumor necrosis factor-mediated pathway are up-regulated. C Up-regulation of isg15 expression in 6 hpf zebrafish embryos by qRT-PCR. D Expression of isg15 in 8-cell stage and High stage embryos after plasmid injection compared with controls. 8 pg, 32 pg, 128 pg: the injected amount of plasmid pEGFP-N1. NC: un-injected zebrafish embryos

Plasmid pEGFP-N1 has core sequences regulating the expression of isg15. A BLASTN alignment of pEGFP-N1 plasmid and isg15 gene sequences (including 2 kbp promoter upstream of TSS) with Kablammo (http://kablammo.wasmuthlab.org/). B Comparison of c-ori, c-CMV and c-HSV (DNAMAN) sequences showing a common 7 bp element, 5ʹ-GTTTGTT-3ʹ. C qRT-PCR results showing that the three mutated fragments of c-ori, c-CMV and c-HSV have weakened activation of isg15 expression in 3 hpf embryos compared with the unmutated fragments. D Responses of HEK 293T cells to transfection with the three DNA fragments and their corresponding mutated sequences. Cells’ responses were significantly weakened after the three sequence mutations. E, F Semi-quantitative PCR and qRT-PCR results showing that the methylation level of H3K4me3 in the isg15 promoter region was significantly increased. MW: DL5000 DNA Molecular Weight Marker

The pEGFP-N1 backbone has sequences that can regulate the expression of isg15. A qRT-PCR results showing that pEGFP-N1, pCMV-VSV-G and pLVX-IRES-ZsGreen1 activate the expression of isg15 in 3 hpf embryos. B Mutation of core plasmid sequences reduces isg15 expression. C qRT-PCR results showing that mutation of core sequences of ori, CMV and HSV in pEGFP-N1 reduces activation of isg15 expression. D Mutation of the core sequence of pCMV-VSV-G and pLVX-IRES-ZsGreen1 weakens responses of HEK 293 T cells. E, F Semi-quantitative PCR and qRT-PCR results showing that the methylation level of H3K4me3 in the isg15 promoter region was significantly increased. MW: DL5000 DNA Molecular Weight Marker

Macrophages recognize the core sequence to allow swallowing and digestion of invading plasmids. A Microinjection of plasmids into the central artery of zebrafish embryos. B qRT-PCR results showing reduced expression of isg15 12 h-post-injection when the core sequences in pEGFP-N1 are mutated. C, D Q-PCR results indicating that pEGFP-N1 was digested more rapidly than all mutant forms of pEGFP-N1. E Confocal scanning image showing macrophages swallowing free DNA fragments in zebrafish

Core sequences protect prokaryotic cells from plasmid infection and mutation of the core sequences increases plasmid transformation efficiency. Transformation efficiencies of different mutant forms of pEGFP-N1 in E. coli DH5α (A), E. coli BL21 (B), and E. coli FastT1 (C). Transformation efficiencies of different mutant forms of pXT7-myh9a in E. coli DH5α (D) and E. coli FastT1 (E). F Transformation efficiencies of different sized forms of pXT7-myh9a with mutant core sequence in ori. When the core sequence in ori is mutated, the number of clones is reduced for the small size pXT7-myh9a but increased for the large plasmid. Sanger sequencing results showing that the ori sequence of pEGFP-N1 favors survival compared with the 5ʹ-GTTTGTT-3ʹ sequence (G), but the ori sequence of pXT7 my17 confers no survival advantage (H)