Novel method for integrating genes at specific sites in mammalian cells via homologous recombination and vectors for accomplishing the same
AssigneeBiogen Idec Inc.
InventorsMitchell R. Reff, Richard Spence Barnett, Karen Retta McLachlan
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Abstract
A method for achieving site specific integration of a desired DNA at a target site in a mammalian cell via homologous recombination is described. This method provides for the reproducible selection of cell lines wherein a desired DNA is integrated at a predetermined transcriptionally active site previously marked with a marker plasmid. The method is particularly suitable for the production of mammalian cell lines which secrete mammalian proteins at high levels, in particular immunoglobulins. Novel vectors and vector combinations for use in the subject cloning method are also provided.
Claims41
A method for inserting a desired DNA at a target site in the genome of a mammalian cell which comprises the following steps:
- (i) transfecting or transforming a mammalian cell with a first plasmid (“marker plasmid”) containing the following sequences:
- (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 mammalian cells which have been successfully integrated with the marker plasmid;
- (ii) selecting a cell which contain the marker plasmid integrated in its genome;
- (iii) transfecting or transforming said selected cell with a second plasmid (“target plasmid”) which contains 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;
- and
- (iv) selecting cells which contain the target plasmid integrated at the target site by screening for the expression of the first selectable marker protein.
A vector system for inserting a desired DNA at a target site in the genome of a mammalian cell which comprises at least the following:
- (i) a first plasmid (“marker plasmid”) containing at least the following sequences:
- (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 mammalian cells which have been successfully integrated with the marker plasmid;
- and
- (ii) a second plasmid (“target plasmid”) 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.
Dependencies are inferred from the claim text.