A 'select and swap' strategy for the isolation of clones with tightly regulated transgenes.
Increasing numbers of biological problems are being addressed by genetic approaches that rely on inducible expression of transgenes. It is desirable that expression of such a transgene is tightly regulated, from close to zero expression in the 'off' state, to appreciable (at least physiological) expression in the 'on' state. Although there are many examples where tight regulation 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 and/or laborious. Here we describe a 'select and swap' strategy for the isolation, from a population of stable transfectants, of clones with tightly regulated transgenes. In this approach, a positively and negatively selectable, inducible marker gene is used to select for clones with optimal transgene regulation. After isolation of such clones, the marker gene is swapped with 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 tetracycline-inducible bacterial gpt gene linked to a promoterless luciferase gene, isolated clones with tight gpt expression and used the Cre/loxP site-specific recombination system to swap the gpt gene with the luciferase gene. We discuss ways for refining and developing the system and widening its applicability.
MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, London, UK.
M J
MJ
Sullivan
A J
AJ
Carpenter
A C
AC
Porter
D015139:Blotting, Southern; D002460:Cell Line; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D005819:Genetic Markers; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D010957:Plasmids; D013752:Tetracycline / Q000378:metabolism; D019076:Transgenes
D005819:Genetic Markers; D007041:Hypoxanthine Phosphoribosyltransferase; D013752:Tetracycline