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

摘要: Increasing numbers of biological problems are being addressed by genetic approaches that rely on inducible expression transgenes. It is desirable such a transgene tightly regulated, from close to zero in the ‘off’ state, appreciable (at least physiological) ‘on’ state. Although there many examples where tight regulation has been achieved, certain factors, including chromosomal position effects due random integration transgene, often cause suboptimal inducibility and make isolation regulated clones difficult and/or laborious. Here we describe ‘select swap’ strategy for isolation, population stable transfectants, with In this approach, positively negatively selectable, marker gene used select optimal regulation. After clones, swapped linked interest use site-specific recombination. To test introduced into human cells plasmid tetracycline-inducible bacterial gpt promoterless luciferase gene, isolated Cre/loxP recombination system swap gene. We discuss ways refining developing widening its applicability.