C - Chemistry – Metallurgy – 12 – N
Patent
C - Chemistry, Metallurgy
12
N
C12N 5/10 (2006.01) A01K 67/027 (2006.01) C12N 15/10 (2006.01) C12N 15/64 (2006.01) C12N 15/85 (2006.01) C12N 15/90 (2006.01)
Patent
CA 2263482
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. 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.
Bode Juergen
Schuebeler Dirk
Seibler Jost
Gesellschaft Fur Biotechnologische Forschung Mbh (gbf)
Macrae & Co.
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