Abstract: The invention relates to a method for marker-free DNA expression cassette exchange in the genome of cells or parts of cells by using the FLP recombinase mediated cassette exchange. A first DNA expression cassette carrying a positive-negative selection marker flanked by two FLP recombinase recognition target (FRT) sites is integrated into a chromosomal locus of the genome. Following selection of cell clones surviving the conditions for positive selection, the first DNA cassette is exchanged by an incoming second DNA expression cassette located on a circular vector and carrying a transgene flanked by the same FRT sites as the first DNA cassette by using FLP-recombinase. The cell clones surviving the conditions for negative selection contain specifically inserted the gene of the incoming DNA cassette without inserted unwanted vector sequences or positive selectable markers.

Abstract: The invention relates to a method for marker-free repetitive DNA expression cassette exchange in the genome of cells or parts of cells by using the FLP recombinase mediated cassette exchange. In a first step a first DNA expression cassette carrying a positive-negative selection marker flanked by a wild type FLP recombinase recognition target (FRT) site on one end and a modified heterospecific FRT on the other end is integrated into a chromosomal locus of the genome for tagging. Following selection of cell clones surviving the conditions for positive selection said first DNA cassette as a second step is exchanged by an incoming second DNA expression cassette located on a circular vector and carrying a homologous or heterologous gene (transgene) of any coding sequence flanked by the same FRT sites as the first DNA cassette by using FLP-recombinase.