Patent US4656134A successfully refreshed from Google Patents.
US4656134A
Expired / Lapsed
US
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Gene amplification in eukaryotic cells
Assignee: Leland Stanford Junior University
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Filing: 1985-04-12
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Granted: 1987-04-07
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Expires: 2005-04-12
Abstract
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.
Inventors
- 👤 Gordon M. Ringold
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 16:54
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85
Claims Analysis
Total Claims: 10
Claim 1
Independent Claim
155 words
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.
Claim 2
Independent Claim
155 words
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.
Claim 3
Dependent Claim
18 words
A method according to claim 1, wherein said DNA becomes integrated into the chromosome of said host cell.
Claim 4
Independent Claim
20 words
A method according any of claims 1 or 2, wherein said DNA sequence of interest codes for a eukaryotic polypeptide.
Claim 5
Dependent Claim
14 words
A method according to claim 3, wherein said polypeptide is foreign to said host.
Claim 6
Independent Claim
48 words
A method according to any of claims 1 or 2, wherein said individual regulatory signals comprise different promoters for each of said amplifiable gene and said DNA sequence of interest, wherein said promoter for said DNA sequence of interest is stronger than the promoter for said amplifiable gene.
Claim 7
Dependent Claim
12 words
A method according to claim 3, wherein said polypeptide is an enzyme.
Claim 8
Dependent Claim
29 words
A method for producing enhanced amounts of a polypeptide, said method comprising: growing in appropriate nutrient medium progeny of a transformant prepared according to the method of claim 3.
Claim 9
Dependent Claim
12 words
A method according to claim 7, wherein said polypeptide is an enzyme.
Claim 10
Dependent Claim
17 words
A method according to claim 1, wherein said amplifiable structural gene codes for the enzyme dihydrofolate reductase.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1990-04-06 | 1990-10-06 | 1991-04-07 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1994-04-06 | 1994-10-06 | 1995-04-07 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1998-04-06 | 1998-10-06 | 1999-04-07 | $7700.00 | Window Closed / Lapsed |