Method for marker-free repetitive DNA expression cassette exchange in the genome of cells or parts of cells

US6992235B2 · kind B2 · utility

Assignee

• Gesellschaft fur Biotechnologische Forschung mbH (GBF) · DE

Inventors

• Jurgen Bode · Kassel, DE
• Jost Seibler · Braunschweig, DE
• Dirk Schübeler · Braunschweig, DE

Key dates

• Filing date: Apr 25, 2001
• Grant date: Jan 31, 2006
• Expiry date: Apr 25, 2021

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12N15/10
• WIPO field: Biotechnology
• WIPO sector: Chemistry

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.