US5122464A
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Method for dominant selection in eucaryotic cells
Assignee: University of Glasgow
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Filing: 1987-01-23
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Granted: 1992-06-16
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Expires: 2007-01-23
Abstract
Abstract PCT No. PCT/GB87/00039 Sec. 371 Date Oct. 23, 1987 Sec. 102(e) Date Oct. 23, 1987 PCT Filed Jan. 23, 1987 PCT Pub. No. WO87/04462 PCT Pub. Date Jul. 30, 1987.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.
Inventors
- 👤 Richard H. Wilson
- 👤 Christopher R. Bebbington
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-03 02:10
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/93; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85; C12N9/14; C12N9/48; C12N9/50; C12N9/64; C12N9/6421; C12N9/6424; C12N9/6456; C12N9/6459; C12Y; C12Y304/00; C12Y304/21; C12Y304/21069
Claims Analysis
Total Claims: 28
Claim 1
Independent Claim
144 words
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.
Claim 2
Independent Claim
144 words
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.
Claim 3
Dependent Claim
36 words
The method of claim 1, wherein step (d) comprises culturing the transformed host cell in media containing a GS inhibitor and selecting for transformant cells which are resistant to progressively increased level of the GS inhibitor.
Claim 4
Dependent Claim
20 words
The method of claim 2, wherein the GS inhibitor is selected from the group consisting of phosphinothricin and methionine sulphoximine.
Claim 5
Dependent Claim
30 words
The method of claim 2 or claim 3, wherein the media containing the GS inhibitor also contain methionine, whereby the concentrations of GS inhibitor in the media can be reduced.
Claim 6
Dependent Claim
24 words
The method of claim 2, wherein the recombinant DNA sequence which encodes an active GS enzyme is under the control of a regulatable promoter.
Claim 7
Dependent Claim
24 words
The method of claim 5, wherein the regulatable promoter is selected from the group consisting of a heat shock promoter and a metallothionein promoter.
Claim 8
Dependent Claim
25 words
The method of claim 5 or claim 6, wherein the regulatable promoter is up-regulated during the culturing and selecting steps and is down-regulated after selection.
Claim 9
Dependent Claim
13 words
The method of claim 1, wherein the desired protein is tissue plasminogen activator.
Claim 10
Independent Claim
155 words
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.
Claim 11
Independent Claim
155 words
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.
Claim 12
Dependent Claim
35 words
The method of claim 9, wherein step (e) comprises culturing the transformed host cell in media containing a GS inhibitor and selecting for transformant cells which are resistant to progressively increased of the GS inhibitor.
Claim 13
Dependent Claim
24 words
The method of claim 10, wherein the recombinant DNA sequence which encodes an active GS enzyme is under the control of a regulatable promoter.
Claim 14
Dependent Claim
24 words
The method of claim 11, wherein the regulatable promoter is selected from the group consisting of a heat shock promoter and a metallothionein promoter.
Claim 15
Dependent Claim
25 words
The method of claim 11 or claim 12, wherein the regulatable promoter is up-regulated during the culturing and selecting steps and is down-regulated after selection.
Claim 16
Dependent Claim
20 words
The method of claim 10, wherein the GS inhibitor is selected from the group consisting of phosphinothricin and methionine sulphoximine.
Claim 17
Dependent Claim
30 words
The method of claim 10 or claim 14, wherein the media containing the GS inhibitor also contain methionine, whereby the concentrations of GS inhibitor in the media can be reduced.
Claim 18
Dependent Claim
13 words
The method of claim 9, wherein the desired protein is tissue plasminogen activator.
Claim 19
Dependent Claim
16 words
The method of claim 1 or claim 9, wherein the host cell is a mammalian cell.
Claim 20
Dependent Claim
16 words
The method of claim 1 or claim 9, wherein the host cell is a CHO-K1 cell.
Claim 21
Independent Claim
108 words
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.
Claim 22
Independent Claim
108 words
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.
Claim 23
Independent Claim
121 words
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.
Claim 24
Independent Claim
121 words
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.
Claim 25
Dependent Claim
9 words
A plasmid including the GS minigene from plasmid pSVLGS.1.
Claim 26
Dependent Claim
9 words
A plasmid including the GS minigene from plasmid pSVLGS.1.
Claim 27
Dependent Claim
10 words
A plasmid including the SV40-GS transcription unit from plasmid pSVLGS.1.
Claim 28
Dependent Claim
10 words
A plasmid including the SV40-GS transcription unit from plasmid pSVLGS.1.
References Cited (Backward Citations)
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