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US 4,656,134 AGranted
Gene amplification in eukaryotic cells
AssigneeThe Board of Trustees of the Leland Stanford Jr. University
InventorGordon M. Ringold
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Abstract
Method and compositions are provided for gene amplification. A DNA unit is prepared capable of replication in a eukaryotic host having tandem genes, where the first gene is capable of complementing an auxotrophic host. The auxotrophic host is transformed with the DNA unit under selective conditions requiring enhanced expression of the first gene. Hosts are then selected for amplification of the first and second genes.
Claims9
A method for obtaining multiple repetitive copies of a DNA sequence of interest comprising a structural gene as continuous repetitive units which comprises:
- transforming a eukaryotic auxotrophic host cell with DNA units, said DNA units comprising:
- (1) an amplifiable structural gene complementing said auxotrophic host cell and capable of amplification upon growth of said host cell under conditions selective for prototrophy;
- (2) joined to said amplifiable structural gene, said DNA sequence of interest;
- and
- (3) individual regulator signals recognized by said host cell for transcription and translation of said structural gene and DNA sequence of interest, to produce transformant cells;
- and
- growing said transformant cells under selective conditions for said amplifiable gene;
- selecting viable cells;
- and
- growing in a nutrient medium said viable cells under increasingly more stringent selective conditions;
- whereby said amplifiable gene and DNA sequence of interest are amplified into multiple copies as continuous repetitive units and are expressed by said host cell.
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