Gene targeting constructs: effects of vector topology on co-expression efficiency of positive and negative selectable...

Targeting of DNA into specific chromosomal loci often involves the use of a negative selectable marker gene to enrich for cell clones that have undergone homologous recombination. In this study we asked if the arrangement of the positive and negative markers in 'knockout' constructs can influence the expression of a lacZ gene that was used as a negative marker. We show that constructs which differ only in their topology have vastly different co-expression efficiencies. The site of DNA linearization was critical. While linearizing at the 5'-end of the lacZ gene was compatible with efficient lacZ expression, linearizing at the 3'-end was always detrimental. We also demonstrate that the topology of the template was more important than the promoter used to drive lacZ expression.