GENE TARGETING CONSTRUCTS - EFFECTS OF VECTOR TOPOLOGY ON COEXPRESSION EFFICIENCY OF POSITIVE AND NEGATIVE SELECTABLE MARKER GENES

Targeting of DNA into specific chromosomal loci often involves the use of a negative selectable marker gene to enrich for cell clones that h ave undergone homologous recombination. In this study we asked if the arrangement of the positive and negative markers in 'knockout' constru cts can influence the expression of a lacZ gene that was used as a neg ative marker. We show that constructs which differ only in their topol ogy have vastly different co-expression efficiencies. The site of DNA linearization was critical. While linearizing at the 5'-end of the lac Z gene was compatible with efficient lacZ expression, linearizing at t he 3'-end was always detrimental. We also demonstrate that the topolog y of the template was more important than the promoter used to drive l acZ expression. (C) 1994 Academic Press, Inc.