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Fig. 3 | BMC Biotechnology

Fig. 3

From: Gene stacking in plant cell using recombinases for gene integration and nucleases for marker gene deletion

Fig. 3

I-SceI-mediated marker excision from site-specific integration (SSI) locus. a The structure of T5-SSI containing I-SceI targets (magenta bars) around Bar gene; b Introduction of I-SceI activity by retransformation of the SSI line, S1, excises Bar gene to generate marker-free SSI; c PCR on UI lines (S1 line retransformed with Ubi:ISceI construct) amplifies ‘excision’ footprints (0.9 or 1.8 kb), indicative of precise excision (#) or indels (*); d Sequence of SSI locus around Bar gene containing loxP upstream and GFP downstream. Red fonts represent I-SceI targets (nick site indicated by ^); e Sequences of the 0.9 kb excision ‘footprints’ obtained from UI lines. Concomitant DSB on the two I-SceI sites followed by joining generates near-perfect repair without the loss of sequences immediately adjacent to the I-SceI targets. The numbers indicate the four repair sequences (1–4) observed in perfect excision lines; f Southern blot analysis of UI lines on KpnI (K)-digested genomic DNA using GFP and Bar probe. Arrow points out the expected excision band (3.5 kb) from near-perfect excision locus. S1, parental SSI line; g PCR analysis of UI lines to detect the presence of Ubi:ISceI gene. Note the absence of the expected 0.8 kb band in UI lines. P, positive control (pUbi:ISceI)

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