US4419446A
Expired / Lapsed
US
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Recombinant DNA process utilizing a papilloma virus DNA as a vector
Assignee: HEALTH AND HUMAN SERVICES United States, THE, Secretary of, Department of
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Filing: 1980-12-31
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Granted: 1983-12-06
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Expires: 2000-12-31
Abstract
Abstract A novel method and composition are provided for the replication and expression of exogenous genes in eukaryotic cells. A segment of a papilloma virus genome capable of extrachromosomal replication is linked to the foreign gene(s) using recombinant DNA techniques to provide a biologically functional replicon with a desired phenotypical property. The replicon is inserted into a eukaryotic cell by transformation, and the isolation of transformant provides cells for replication and expression of the DNA molecules present in the modified plasmid. The transforming region of the bovine papilloma virus provides a unique vector in that it provides both the capability of autonomous extrachromosomal replication but also the malignant transformed phenotype. Thus, genes which of themselves provide no selectable phenotypical property can be conveniently and efficiently introduced into eukaryotic cells and the transformants selected. The method is useful in that the foreign DNA is faithfully expressed and the gene products (proteins), such as pro-insulin (an insulin precursor) is synthesized.
Inventors
- 👤 Peter M. Howley
- 👤 Nava Sarver
- 👤 Ming-Fan Law
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 16:05
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85; C12N15/86; C12N2710/00; C12N2710/00011; C12N2710/20011; C12N2710/20041; C12N2710/20043
Claims Analysis
Total Claims: 9
Claim 1
Independent Claim
123 words
A process for producing a recombinant DNA suitable for introduction and replication in eukaryotic cells comprising the steps of: (a) cleaving bovine papilloma virus (BPV) type 1 or type 2 to produce a first linearized DNA segment selected from the group consisting of BPV-1 DNA cleaved at Bam HI; BPV-1 DNA cleaved at Hind III; BPV-2 DNA cleaved at Hind III; and the 69% subgenomic fragment; BPV 69T DNA, cleaved from BPV-1 DNA at Bam HI and Hind III, (b) combining said first linear DNA segment with a second linear DNA segment containing a foreign gene and having termini ligatable to the termini of the first segment under joining conditions to join said first and second segments to provide a functional recombinant DNA.
Claim 2
Dependent Claim
123 words
A process for producing a recombinant DNA suitable for introduction and replication in eukaryotic cells comprising the steps of: (a) cleaving bovine papilloma virus (BPV) type 1 or type 2 to produce a first linearized DNA segment selected from the group consisting of BPV-1 DNA cleaved at Bam HI; BPV-1 DNA cleaved at Hind III; BPV-2 DNA cleaved at Hind III; and the 69% subgenomic fragment; BPV 69T DNA, cleaved from BPV-1 DNA at Bam HI and Hind III, (b) combining said first linear DNA segment with a second linear DNA segment containing a foreign gene and having termini ligatable to the termini of the first segment under joining conditions to join said first and second segments to provide a functional recombinant DNA.
Claim 3
Dependent Claim
133 words
A process for persistent foreign gene replication in eukaryotic cells comprising the steps of: (a) cleaving bovine papilloma virus (BPV) type 1 or type 2 to produce a first linearized DNA segment selected from the group consisting of BPA-1 DNA cleaved at Bam HI; BPV-1 DNA cleaved at Hind III; BPV-2 DNA cleaved at Hind III and the 69% subgenomic fragment; BPV 69T DNA, cleaved from BPV-1 DNA at Bam HI and Hind III, (b) combining said first linear DNA segment and a second linear segment containing a foreign gene to form a recombinant DNA; (c) introducing the recombinant DNA into eukaryotic cells in the presence of calcium chloride to produce transformants of cells; (d) growing transformants under appropriate nutrient conditions until transformed foci appear; (e) amplifying individual foci to desired mass culture.
Claim 4
Dependent Claim
133 words
A process for persistent foreign gene replication in eukaryotic cells comprising the steps of: (a) cleaving bovine papilloma virus (BPV) type 1 or type 2 to produce a first linearized DNA segment selected from the group consisting of BPA-1 DNA cleaved at Bam HI; BPV-1 DNA cleaved at Hind III; BPV-2 DNA cleaved at Hind III and the 69% subgenomic fragment; BPV 69T DNA, cleaved from BPV-1 DNA at Bam HI and Hind III, (b) combining said first linear DNA segment and a second linear segment containing a foreign gene to form a recombinant DNA; (c) introducing the recombinant DNA into eukaryotic cells in the presence of calcium chloride to produce transformants of cells; (d) growing transformants under appropriate nutrient conditions until transformed foci appear; (e) amplifying individual foci to desired mass culture.
Claim 5
Dependent Claim
37 words
A Eukaryotic cloning vector consisting essentially of the subgenomic fragment BP V 69T consisting of the 69 percent transforming region of bovine papilloma virus type 1 (BPV-1) cleaved from BPV-1 DNA at Bam HI and Hind III.
Claim 6
Dependent Claim
37 words
A Eukaryotic cloning vector consisting essentially of the subgenomic fragment BP V 69T consisting of the 69 percent transforming region of bovine papilloma virus type 1 (BPV-1) cleaved from BPV-1 DNA at Bam HI and Hind III.
Claim 7
Dependent Claim
16 words
A recombinant DNA plasmid consisting essentially of the cloning vector of claim 3 and foreign DNA.
Claim 8
Dependent Claim
15 words
The recombinant DNA plasmid of claim 4 wherein the foreign DNA is eukaryotic or procoryotic.
Claim 9
Dependent Claim
30 words
A method of producing a protein foreign to an eukaryotic cell which comprises conducting the process of claim 2 until the foreign gene produces protein and then isolating the protein.
References Cited (Backward Citations)
US4237224A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1986-12-05 | 1987-06-06 | 1987-12-06 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1990-12-05 | 1991-06-06 | 1991-12-06 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1994-12-05 | 1995-06-06 | 1995-12-06 | $7700.00 | Window Closed / Lapsed |