Generation of an artificial ring chromosome in Arabidopsis by Cre/LoxP-mediated recombination

A eukaryotic chromosome consists of a centromere, two telomeres and a number of replication origins, and 'artificial chromosomes' may be created in yeast and mammals when these three elements are artificially joined and introduced into cells. Plant artificial chromosomes (PACs) have been suggested as new vectors for the development of new crops and as tools for basic research on chromosomes. However, indisputable PAC formation has not yet been confirmed. Here, we present a method for generating PACs in the model plant Arabidopsis thaliana using the Cre/LoxP and Activator/Dissociation element systems. The successfully generated PAC, designated AtARC1 (A. thaliana artificial ring chromosome 1), originated from a centromeric edge of the long arm of chromosome 2, but its size (2.85 Mb) is much smaller than that of the original chromosome (26.3 Mb). Although AtARC1 contains only a short centromere domain consisting of 180 bp repeats approximately 250 kb in length, compared with the 3 Mb domain on the original chromosome 2, centromere-specific histone H3 (HTR12) was detected on the centromeric region. This result supported the observed stability of the PAC during mitosis in the absence of selection, and transmission of the PAC to the next generation through meiosis. Because AtARC1 contains a unique LoxP site driven by the CaMV 35S promoter, it is possible to introduce a selectable marker and desired transgenes into AtARC1 at the LoxP site using Cre recombinase. Therefore, AtARC1 meets the criteria for a PAC and is a promising vector.