Patent ID: 6413777
Filing Date: 2002-07-02
Classification: C07K,C12N

Abstract:
A eukaryotic cell which comprises a desired DNA integrated at a target site in its gene which has been transfected or transformed with at least the following:(i) a first plasmid (â€œmarker plasmidâ€ ) containing at least the following (a) a region of DNA that is heterologous to the mammalian cell genome which when integrated in the mammalian cell genome provides a unique site for homologous recombination; (b) a DNA fragment encoding a portion of a first selectable marker protein; and (c) at least one other selectable marker DNA that provides for selection of eukaryotic cells which have been successfully integrated with the marker plasmid; and (ii) a second plasmid (â€œtarget plasmidâ€ ) which contains at least the following sequences: (a) a region of DNA that is identical or is sufficiently homologous to the unique region in the marker plasmid such that this region of DNA can recombine with said DNA via homologous recombination; (b) a DNA fragment encoding a portion of the same selectable marker contained in the marker plasmid, wherein the active selectable marker protein encoded by said DNA is only produced if said fragment is expressed in association with the fragment of said selectable marker DNA contained in the marker plasmid.