[A simple and visible assay for cre recombinase activity in Escherichia coli by using two incompatible plasmids].

The Cre/loxP system derived from bacteriophage P1 is widely used to carry out complex manipulations of DNA molecules both in vitro and in vivo. In order to further characterize and modify the Cre/loxP system, a convenient method for assaying the recombination efficiency is needed. A simple and visible assay is described, in which two incompatible plasmids, separately carrying the cre gene and loxP-flanked gfp gene, were co-transferred into E. coli. The cre gene was inserted into a kanamycin-resistant bacterial expression vector, designated pET30a-Cre. The gfp gene, flanked by directly repeated loxP sites, was cloned into an ampicillin-resistant expression vector to generate pET23b-loxGFP. E. coli BL21 (DE3) was cotransformed with pET30a-Cre and pET23b-loxGFP, and cultured in the presence of both ampicillin and kanamycin. Under UV illumination, the Cre-mediated recombination events can be easily detected. The fidelity of recombination was verified by SDS-PAGE analysis and restriction analysis followed by DNA sequencing. Thus, this cotransformation method provides a straightforward assay that can be used to modify the Cre/loxP system.

[Using green fluorescent protein as a reporter to monitor elimination of selectable marker genes from transgenic plants].

In genetic modification of plants, once the transformants are obtained, selection markers are no longer required in mature plants. At present, the Cre/lox site-specific recombination system is most widely used to eliminate the selectable marker genes from the transgenic plants. In this study, attempt was made to favour the selection of marker-free plants in the re-transformation method. Green fluorescent protein (GFP) can be directly visualized in living cells, tissues or organisms under UV illumination. This advantage of GFP is exploited in the development of a practical approach in which GFP is used as a visual marker to monitor the removal of the selectable marker gene from transgenic plants. For that purpose, the pGNG binary vector was constructed, in which the GFP gene (gfp) was linked to the expression cassette Nos P-nptII-NosT and the two units were cloned between two directly-orientated lox sites. The CaMV 35S promoter was placed before the first lox site and used to drive GFP expression. The beta-glucuronidase gene (gus) of Escherichia coli was cloned behind the second lox site without a promoter, thus would not be expressed in this position. Tobacco plants were first transformed with pGNG and selected on kanamycin (Kan)-containing media. Regenerated transgenic shoots were readily singled out by GFP fluorescence. The GFP-expressing plants were then re-transformed with pCambia1300-Cre containing hygromycin phosphotransferase gene (hpt) as a selectable marker gene. The Cre-mediated recombination resulted in the elimination of lox-flanked genes, herein gfp and nptII, from the plant genome and brought the GUS gene next to the 35S promoter. Our data demonstrated that transgenic plants free of nptII were easily selected by monitoring the loss of green fluorescence, and at the same time, GUS (here as a target protein) was expressed in the nptII-free plants. Finally, hpt and cre were removed from the progenies of the nptII-free plants by gene segregation.