Cre-loxP mediated marker elimination and gene reactivation at the waxy locus created in rice genome based on strong positive–negative selection
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概要
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The combination of homologous and site-specific recombination, if possible, could create mutants, such as tissue- or growth phase- specific gene knockout. In higher plants, there is no information on the site-specific recombination of the endogenous gene locus modified by gene targeting (GT) because of still limited success of reproducible GT for the endogenous gene. Our method of rice GT mediated by a strong positive–negative selection is appropriate for combined use with site-specific recombination because, in our method, it is possible to manipulate genome sequence variously without any unintentional random transformation, including ectopic targeting. We had previously created a targeted-waxy mutant by inserting the hygromycin phospho transferase (hpt) with ΔEn (a functional transcriptional stop sequence from En/Spm transposon), into the Waxy intron 1 region upstream of the translational initiation site. Since this inserted sequence was sandwiched by a pair of loxP in the same orientation, here, we tested Cre-loxP mediated elimination of this targeted insertion. The Cre was transiently expressed by a highly active promoter of CaMV 35S with the castor bean catalase intron 1 in the calli of the targeted-waxy homozygote. Cells derived from the treated calli were grown independently and applied to PCR screening as a bulk of calli clumps. Although the efficiency was quite low, Cre-loxP mediated hpt-ΔEn elimination and Waxy reactivation in the pollen of regenerated plants were detected as expected in some cells.