Rare instances of Cre-mediated deletion product maintained in transgenic wheat

Previously, we described a Cre-lox based strategy to convert a complex multi-copy integration pattern to a single-copy transgene (Srivastava et al., 1999). When a lox-containing transgenic line of wheat was crossed with a cre-expressing line, extra copies of the transgene were deleted by site-specific recombination. This process included the removal of a lox-flanked selection marker gene, bar. Three out of six F₁ plants were chimeric for the resolved and the complex loci because both completely resolved and incompletely resolved patterns were found in the F₂ population. From one F₁ plant, 4 out of 20 F₂ progeny showed not only incomplete resolution of the complex integration pattern, but also the presence of a circular loxP-bar-nos3 fragment, which we refer to as the bar circle. This bar circle was detected in subsequent generations, and was associated with the presence of both the lox transgene and the cre locus. We hypothesize that the cre gene in these bar circle plants must have undergone a genetic or epigenetic change that altered the spatial and/or temporal pattern of cre expression. Late expression might excise the DNA incompletely, and late in development. What is surprising is that the DNA is not degraded, but remains in the cells as an extra-chromosomal circular molecule.