Dosage-dependent gene expression from direct repeat locus in rice developed by site-specific gene integration.

In the standard plant transformation practice, transgene copy number is often inversely correlated with transgene expression. As the integration locus generated by standard methods is mostly complex, consisting of both full-length and partial copies arranged in direct or inverted repeat configurations, it is difficult to parse the effect of copy number and locus structure. To clearly study the effect of transgene copy number on gene expression, it is important to control the locus structure and integrate full-length copies. In this study, the effect of transgene copy number on transgene expression in plant cells was determined using rice callus as a model. To generate full-length integrations, Cre-lox-mediated site-specific gene integration method was used. Transgenic rice lines consisting of one to three copies of beta-glucuronidase or green fluorescent protein genes were developed. Site-specific integration lines were characterized and subjected to expression analysis. Lines containing two or three copies of either reporter genes displayed 2-4 times higher expression compared to the single-copy lines. Therefore, dosage-dependent transgene expression can be obtained by integrating full-length copies, and site-specific gene integration approach can serve as an efficient tool for generating precise multi-copy integrations.