Methods for constructing clones for protein expression in mammalian cells.

Multisite Gateway technology is a DNA cloning method based on in vitro site-specific recombination that is becoming increasingly popular because it allows quick and highly efficient assembly of multiple DNA fragments into a vector backbone. In the conventional Gateway Multisite strategy, cloning of multiple DNA fragments requires recombination of multiple entry clones with a single destination vector. The -limitation of this approach is that as the number of entry clones increases, the efficiency of the assembly reactions decreases due to difficulty in successfully recognizing multiple pairs of matched att signals simultaneously. To address this problem, we have devised methods to generate modular expression clones, modular entry clones, and modular destination vectors. These allow many DNA fragments to be -assembled stepwise into complex expression clones. We describe here how to construct these intermediate clones and vectors, and how to use these modules to construct expression clones comprising ten or more DNA -segments. These principles can be applied to make multicomponent DNAs for many applications.