Gene disruption by targeted integration of transfected constructs becomes increasingly popular for studies of gene function. The chicken B cell line DT40 has been widely used as a model for gene knock-outs due to its high targeted integration activity. Disruption of multiple genes and complementation of the phenotypes is, however, restricted by the number of available selectable marker genes. It is therefore highly desirable to recycle the selectable markers using a site-specific recombination system like Cre/loxP. We constructed three plasmid vectors (neoR, puroR and bsr), which carry selectable marker genes flanked by two different mutant loxP sites. After stable transfection, the marker genes can be excised from the genome by transient induction of Cre recombinase expression. This excision converts the two mutant loxP sites to an inactive double-mutant loxP. Furthermore we constructed a versatile expression vector to clone cDNA expression cassettes between mutant loxP sites. This vector can also be used to design knock-out constructs in which the floxed marker gene is combined with a cDNA expression cassette. This construct enables gene knock-out and complementation in a single step. Gene expression can subsequently be terminated by the Cre mediated deletion of the cDNA expression cassette. This strategy is powerful for analyzing essential genes, whose disruption brings lethality to the mutant cell. Mutant loxP vectors have been developed for the recycle of selectable markers and conditional gene knock-out approaches. As the marker and the cDNA expression cassettes are driven by the universally active and evolutionary conserved β-actin promoter, they can be used for the selection of stable transfectants in a wide range of cell lines.
