Patent US4436815A successfully refreshed from Google Patents.
US4436815A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Method for stabilizing and selecting recombinant DNA containing host cells
Assignee: Eli Lilly and Co
•
Filing: 1981-11-27
•
Granted: 1984-03-13
•
Expires: 2001-11-27
Abstract
Abstract An improved method for stabilizing and selecting host cells containing recombinant DNA which expresses a functional polypeptide and the novel organisms and cloning vectors for the practice thereof.
Inventors
- 👤 Charles L. Hershberger
- 👤 Paul R. Rosteck, Jr.
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 23:41
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/635; C07; C07K; C07K14/00; C07K14/435; C07K14/575; C07K14/57581; C07K14/62; C12N15/70; Y; Y10; Y10S; Y10S930/00; Y10S930/01; Y10S930/18
Claims Analysis
Total Claims: 29
Claim 1
Independent Claim
198 words
In the method for stabilizing and selecting host cells containing recombinant DNA in which host cells are transformed with a recombinant DNA cloning vector which contains both the ˜2.5 kb BglII cI repressor-containing restriction fragment of bacteriophage λ and a gene which expresses a functional polypeptide, and in which the transformed host cells are lysogenized with a lysogenic organism containing a marker which is lethal or conditionally lethal in the host cells but which is repressed in the transformed host cell by the repressor gene contained in the recombinant DNA cloning vector, an improvement wherein the improvement comprises: transforming the host cells with a recombinant DNA cloning vector comprising (a) the PstI-HincII cI repressor-containing restriction fragment of bacteriophage λ; and (b) a gene which expresses a functional polypeptide; subject to the limitation that the host cells are E. coli, the lysogenic organism is bacteriophage λ and the recombinant DNA cloning vector contains a replicon and a promoter which are not sensitive to the repressor, and subject to the further limitation, that when the transformed host cells are lysogenized with a lysogenic organism containing a gene which is conditionally lethal, the resulting host cells are cultured under restrictive conditions.
Claim 2
Dependent Claim
198 words
In the method for stabilizing and selecting host cells containing recombinant DNA in which host cells are transformed with a recombinant DNA cloning vector which contains both the ˜2.5 kb BglII cI repressor-containing restriction fragment of bacteriophage λ and a gene which expresses a functional polypeptide, and in which the transformed host cells are lysogenized with a lysogenic organism containing a marker which is lethal or conditionally lethal in the host cells but which is repressed in the transformed host cell by the repressor gene contained in the recombinant DNA cloning vector, an improvement wherein the improvement comprises: transforming the host cells with a recombinant DNA cloning vector comprising (a) the PstI-HincII cI repressor-containing restriction fragment of bacteriophage λ; and (b) a gene which expresses a functional polypeptide; subject to the limitation that the host cells are E. coli, the lysogenic organism is bacteriophage λ and the recombinant DNA cloning vector contains a replicon and a promoter which are not sensitive to the repressor, and subject to the further limitation, that when the transformed host cells are lysogenized with a lysogenic organism containing a gene which is conditionally lethal, the resulting host cells are cultured under restrictive conditions.
Claim 3
Dependent Claim
15 words
The method of claim 1 in which the recombinant DNA cloning vector is a plasmid.
Claim 4
Dependent Claim
15 words
The method of claim 1 in which the recombinant DNA cloning vector is a bacteriophage.
Claim 5
Dependent Claim
45 words
The method of claim 1 in which the gene which expresses a functional polypeptide is selected from the group of genes consisting of naturally occurring genes, non-naturally occurring genes, and genes which are in part naturally occurring and are in part synthetic or non-naturally occurring.
Claim 6
Dependent Claim
59 words
The method of claim 1 in which the gene which expresses a functional polypeptide is selected from the group consisting of genes coding for human insulin, human pre-proinsulin, human proinsulin, human insulin A-chain, human insulin B-chain, non-human insulin, human growth hormone, non-human growth hormone, human interferon, non-human interferon, viral antigen, urokinase, any polypeptide, and any peptide hormone or enzyme.
Claim 7
Dependent Claim
12 words
The method of claim 1 in which the cI repressor is cI857.
Claim 8
Dependent Claim
23 words
The method of claim 1 in which the lysogenic organism contains a bacteriophage λcI gene which does not produce a functional cI repressor.
Claim 9
Dependent Claim
14 words
The method of claim 7 in which the lysogenic organism is bacteriophage lambda cI90.
Claim 10
Dependent Claim
13 words
The method of claim 1 in which the lysogenic organism is bacteriophage λcI857.
Claim 11
Dependent Claim
15 words
The method of claim 1 in which the lysogenic organism has the cI gene deleted.
Claim 12
Dependent Claim
13 words
The method of claim 1 in which the host cells are E. coli.
Claim 13
Dependent Claim
14 words
The method of claim 11 in which the host cells are E. coli K12.
Claim 14
Dependent Claim
15 words
The method of claim 1 in which the recombinant DNA cloning vector is plasmid pPR17.
Claim 15
Dependent Claim
15 words
The method of claim 1 in which the recombinant DNA cloning vector is plasmid pPR18.
Claim 16
Dependent Claim
13 words
A recombinant DNA cloning vector which is a cloning vector of claim 1.
Claim 17
Dependent Claim
12 words
The recombinant DNA cloning vector of claim 15 which is plasmid pPR17.
Claim 18
Dependent Claim
12 words
The recombinant DNA cloning vector of claim 15 which is plasmid pPR18.
Claim 19
Dependent Claim
15 words
A host cell which is transformed with a recombinant DNA cloning vector of claim 15.
Claim 20
Dependent Claim
11 words
The transformed host cell of claim 18 which is E. coli.
Claim 21
Dependent Claim
18 words
The transformed lysogenized host cell of claim 1 which is E. coli K12 C600R k -M k -λcI90/pPR17.
Claim 22
Dependent Claim
14 words
The transformed lysogenized host cell of claim 1 which is E. coli K12 294λcI90/pPR18.
Claim 23
Dependent Claim
14 words
The transformed lysogenized host cell of claim 1 which is E. coli K12 RV308λcI90/pPR17.
Claim 24
Dependent Claim
14 words
The transformed lysogenized host cell of claim 1 which is E. coli K12 RV308λcI90/pPR18.
Claim 25
Dependent Claim
12 words
The recombinant DNA cloning vector of claim 15 which is plasmid pPR12.
Claim 26
Dependent Claim
11 words
A host cell transformed with the cloning vector of claim 24.
Claim 27
Dependent Claim
17 words
The transformed host cell of claim 25 which is E. coli K12 C600R k -M k -λcI90/pPR12.
Claim 28
Dependent Claim
4 words
The restriction fragment pPR12Δ2.
Claim 29
Dependent Claim
4 words
The restriction fragment pPR12Δ2.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1987-03-13 | 1987-09-12 | 1988-03-13 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1991-03-13 | 1991-09-12 | 1992-03-13 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1995-03-13 | 1995-09-12 | 1996-03-13 | $7700.00 | Window Closed / Lapsed |