A 'select and swap' strategy for the isolation of clones with tightly regulated transgenes

Increasing numbers of biological problems are being addressed by genetic ap proaches that rely on inducible expression of transgenes. It is desirable t hat expression of such a transgene is tightly regulated, from close to zero expression in the 'off' state, to appreciable (at least physiological) exp ression in the 'on' state. Although there are many examples where tight reg ulation has been achieved, certain factors, including chromosomal position effects due to random integration of the transgene, often cause suboptimal inducibility and make the isolation of tightly regulated clones difficult a nd/or laborious. Here we describe a 'select and swap' strategy for the isol ation, from a population of stable transfectants, of clones with tightly re gulated transgenes. In this approach, a positively and negatively selectabl e, inducible marker gene is used to select for clones with optimal transgen e regulation. After isolation of such clones, the marker gene is swapped wi th a linked gene of interest by the use of site-specific recombination. To test this strategy we introduced into human cells a plasmid with a tetracyc line-inducible bacterial gpt gene linked to a promoterless luciferase gene, isolated clones with tight gpt expression and used the Cre/loxP site-speci fic recombination system to swap the gpt gene with the luciferase gene. We discuss ways for refining and developing the system and widening its applic ability.