Chemically induced marker removal

The marker removal system (schemes in Figures 1 & 2) fits easily in existing Agrobacterium -mediated transformation protocols.

Figure 1. Selection scheme for producing marker-free transgenic plants using pMF1. A similar selection scheme can be followed using pMF2 or pMF3 vectors, when positive selection on either hygromycin or phosphinothricin, respectively, is preferred.
Figure 1. Selection scheme for producing marker-free transgenic plants using pMF1. A similar selection scheme can be followed using pMF2 or pMF3 vectors, when positive selection on either hygromycin or phosphinothricin, respectively, is preferred.

After positive selection of transgenic tissue or plants, chemical induction of Recombinase R activity will result in elimination of DNA-sequences that are flanked by intact recombination sites (Rs). The Recombinase R protein is inactivated by the presence of the Ligand Binding Domain (LBD) of the glucocorticoid receptor. Activation of the Recombinase R protein activity is achieved by an overnight incubation of plant tissue in a 10 ┬ÁM dexamethasone solution. Subsequent (negative) selection on 5-FC should prevent development of plant tissue still containing the cod A-gene, thereby preventing the occurrence of chimeras due to incomplete DNA excision.

Figure 2. Schematic representation depicting the T-DNA of pMF1 before and after Recombinase R-mediated removal of the DNA-sequences that are flanked by the recombination sites (Rs). Black arrows indicate primer locations for PCR-check for confirmation of the recombination event.
Figure 2. Schematic representation depicting the T-DNA of pMF1 before and after Recombinase R-mediated removal of the DNA-sequences that are flanked by the recombination sites (Rs). Black arrows indicate primer locations for PCR-check for confirmation of the recombination event.