Method for dominant selection in eucaryotic cells
AssigneesCelltech Limited, a British Company, The University Court of the University of Glasgow
InventorsRichard H. Wilson, Christopher R. Bebbington
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Abstract
Recombinant DNA sequences which encode the complete amino acid sequence of a glutamine synthetase, vectors containing such sequences, and methods for their use, in particular as dominant selectable markers, for use in co-amplificiation of non-selected genes and in transforming host cell lines to glutamine independence.
Claims22
A method for co-amplifying a recombinant DNA sequence which encodes the complete amino acid sequence of a desired protein other than a glutamine synthetase (GS), comprising:
- (a) providing an expression vector comprising a recombinant DNA sequence which encodes an active GS enzyme and the recombinant DNA sequence which encodes the complete amino acid sequence of the desired protein other than GS;
- (b) providing a eukaryotic host cell which is a GS prototroph;
- (c) transforming said host cell with said expression vector;
- and
- (d) culturing said transformed host cell under conditions which allow transformants containing an amplified number of copies of the vector-derived recombinant DNA sequence which encodes an active GS enzyme to be selected wherein said transformants also contain an amplified number of copies of the desired recombinant DNA sequence which encodes the complete amino acid sequence of the desired protein other than GS.
A method for co-amplifying a recombinant DNA sequence which encodes the complete amino acid sequence of a desired protein other than a GS, comprising:
- (a) providing a first expression vector comprising a recombinant DNA sequence which encodes an active GS enzyme;
- (b) providing a second expression vector comprising the recombinant DNA sequence which encodes the complete amino acid sequence of the desired protein other than GS;
- (c) providing a eukaryotic host cell which is a CS prototroph;
- (d) transforming said host cell with both said first and said second expression vectors;
- and
- (e) culturing said transformed host cell under conditions which allow transformants containing an amplified number of copies of the first expression vector-derived recombinant DNA sequence which encodes an active GS enzyme to be selected, wherein said transformants also contain an amplified number of copies of the desired recombinant DNA sequence which encodes the complete amino acid sequence of a protein other than GS.
A method for using a vector as a dominant selectable marker in a cotransformation process comprising:
- (a) providing an expression vector comprising a recombinant DNA sequence which encodes an active GS enzyme and a recombinant DNA sequence which encodes the complete amino acid sequence of a desired protein other than GS;
- (b) providing a eukaryotic host cell which is a GS prototroph;
- (c) transforming the host cell with the expression vector;
- and
- (d) selecting transformant cells which are resistant to GS inhibitors, whereby transformant cells are selected in which the vector-derived recombinant DNA sequence which encodes an active GS enzyme serves as a dominant selectable and co-amplifiable marker.
A method for using a vector as a dominant selectable marker in a cotransformation process comprising:
- (a) providing a first expression vector comprising a recombinant DNA sequence which encodes an active GS enzyme;
- (b) providing a second expression vector comprising a recombinant DNA sequence which encodes the complete amino acid sequence of a desired protein other than GS;
- (c) providing a eukaryotic host cell which is a GS prototroph;
- (d) transforming said host cell with both said first and second expression vectors;
- and
- (e) selecting transformant cells which are resistant to GS inhibitors, whereby transformant cells are selected in which the first expression vector-derived recombinant DNA sequence which encodes an active GS enzyme serves as a dominant selectable and co-amplifiable marker.
A plasmid including the GS minigene from plasmid pSVLGS.1.
A plasmid including the SV40-GS transcription unit from plasmid pSVLGS.1.
Dependencies are inferred from the claim text.