Submitted to: Plant Molecular Biology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 12/6/2002
Publication Date: 2/7/2003
Citation: SRIVASTAVA, V., OW, D.W. RARE INSTANCES OF CRE-MEDIATED DELETION PROUDCT MAINAINED IN TRANSGENIC WHEAT. PLANT MOLECULAR BIOLOGY 52:661-668. 2003. Interpretive Summary: This article describes a rare event observed with Cre-lox site-specific recombination. The excision product is detected in the plant genome as a free circular DNA. It is generated de novo in each generation from a particular cre allele that appears to have mutated, probably through epigenetic modification, as its DNA is hypermethylated. This shows that in some rare instances, it is possible to obtain plants where the deleted DNA is not lost, but remains in the plant genome as an extrachromosomal element.
Technical Abstract: 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.