CRE LOX-MEDIATED SITE-SPECIFIC INTEGRATION OF AGROBACTERIUM T-DNA IN ARABIDOPSIS-THALIANA BY TRANSIENT EXPRESSION OF CRE/

The Cre/lox system was used to obtain targeted integration of an Agrob acterium T-DNA at a lox site in the genome of Arabidopsis thaliana. Si te-specific recombinants, and not random events, were preferentially s elected by activation of a silent lox-neomycin phosphotransferase (npt II) target gene. To analyse the effectiveness of Agrobacterium-mediate d transfer we used T-DNA Vectors harbouring a single lex sequence (thi s vector had to circularize at the T-DNA left- and right-border sequen ces prior to site-specific integration) or two lex sequences (this vec tor allowed circularization at the lex sequences within the T-DNA eith er prior to or after random integration, followed by targeting of the circularized vector), respectively. Furthermore, to control the revers ibility of the integration reaction, Cre recombinase was provided tran siently by using a cotransformation approach. One precise stable integ rant was found amongst the recombinant calli obtained after transforma tion with a double-lex T-DNA vector. The results indicate that Agrobac terium-mediated transformation can be used as a tool to obtain site-sp ecific integration.