Efficient linking and transfer of multiple genes by a multigene assembly and transformation vector system

Introduction and expression of multiple transgenes is frequently required for basic and applied studies. However, at present, multigene transformation is very difficult due to technical limitations of existing methods. Here,we describe a vector system for efficient multigene assembly and transformation. The system consists of a transformation-competent artificial chromosome (TAC)-based acceptor vector together with two donor vectors. By exploiting the Cre/IoxP recombination system and homing endonucleases, multiple rounds of gene assembly cycling were carried out with alternate use of the donor vectors, and multiple genes were sequentially delivered into the TAC vector. With this system, we created constructs containing as many as 10 foreign DNA fragments. Multiple genes, including six resistant genes stacked in a construct, were transferred into rice genome by Agrobacterium-mediated transformation. This system extends the repertoire of molecular genetic studies and biotechnological endeavors by enabling simultaneous manipulation of multiple genes.