EP0117060A2
Expired / Lapsed
EP
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Methods of screening and amplification in eukaryotic host cells, and nucleotide sequences and expression vectors for use therein
Assignee: Genentech Inc
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Filing: 1984-01-18
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Granted: —
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Expires: 2004-01-18
Abstract
Abstract A plasmid which comprises a DNA sequence coding for a DHFR protein with a low binding affinity for MTX. Also, plasmids which contain the foregoing sequence along with a sequence encoding a desired heterologous protein, each sequence operably linked to a sequence capable of effecting its expression. Methods of transforming eukaryotic cells with the genetic sequences for a desired protein, selecting the successful transformants, and amplifying in the transformants, by employing the sequence coding for DHFR protein with low MTX binding affinity. Wild type eukaryotic cells may be used as hosts; it is not necessary to use mutants which have become deficient in a particular enzyme or cofactor.
Inventors
- 👤 Arthur David Levinson
- 👤 Christian Clinton Simonsen
- 👤 Elizabeth Mcleod Yelverton
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-24 18:30
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/005; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85; C12N9/00; C12N9/0004; C12N9/0012; C12N9/0026; C12N9/0028; C12N9/14; C12N9/48; C12N9/50; C12N9/64; C12N9/6421; C12N9/6424; C12N9/6456; C12N9/6459; C12Y; C12Y304/00; C12Y304/21; C12Y304/21069; C12N2730/00; C12N2730/00011; C12N2730/10011; C12N2730/10111; C12N2730/10122
Claims Analysis
Total Claims: 31
Claim 1
Independent Claim
15 words
A nucleotide sequence of the structure substantially as is set forth in Figure lb hereof.
Claim 2
Dependent Claim
15 words
A nucleotide sequence of the structure substantially as is set forth in Figure lb hereof.
Claim 3
Dependent Claim
20 words
A nucleotide sequence coding for the amino acid sequence of DHFR 3T6-R400, substantially free of impurities and of intron sequences.
Claim 4
Dependent Claim
20 words
A nucleotide sequence coding for the amino acid sequence of DHFR 3T6-R400, substantially free of impurities and of intron sequences.
Claim 5
Dependent Claim
36 words
An expression vector which comprises the DNA sequence coding for the amino acid sequence of a DHFR protein with a low binding affinity for MTX operably linked to a DNA sequence capable of effecting expression thereof.
Claim 6
Dependent Claim
36 words
An expression vector which comprises the DNA sequence coding for the amino acid sequence of a DHFR protein with a low binding affinity for MTX operably linked to a DNA sequence capable of effecting expression thereof.
Claim 7
Dependent Claim
21 words
A replicable plasmid capable, in a transformant eukaryotic host cell, of expressing DHFR protein with a low binding affinity for MTX.
Claim 8
Dependent Claim
21 words
A replicable plasmid capable, in a transformant eukaryotic host cell, of expressing DHFR protein with a low binding affinity for MTX.
Claim 9
Dependent Claim
14 words
The plasmid of claims 3 or 4 wherein the DHFR protein is DHFR 3T6-R400.
Claim 10
Dependent Claim
3 words
The plasmid pFR400.
Claim 11
Dependent Claim
3 words
The plasmid pFR400.
Claim 12
Dependent Claim
16 words
A eukaryotic cell line transformed with the vector of any one of claims 3 to 6.
Claim 13
Dependent Claim
53 words
An expression vector which comprises a first DNA sequence encoding a DHFR protein with a low binding affinity for MTX and a second DNA sequence encoding a desired heterologous protein, each of said first and second sequences being operably linked to a DNA sequence capable of effecting expression of the respective coding sequence.
Claim 14
Dependent Claim
53 words
An expression vector which comprises a first DNA sequence encoding a DHFR protein with a low binding affinity for MTX and a second DNA sequence encoding a desired heterologous protein, each of said first and second sequences being operably linked to a DNA sequence capable of effecting expression of the respective coding sequence.
Claim 15
Dependent Claim
30 words
The vector of claim 8 wherein the first and second D N A sequences are operably linked to a single DNA sequence capable of effecting expression of both said sequences.
Claim 16
Dependent Claim
14 words
The vector of claim 8 or 9 wherein the DHFR protein is DHFR 3T6-R400.
Claim 17
Dependent Claim
17 words
The vector of claim 8, 9 or 10 wherein the desired heterologous protein is HBsAg or tPA.
Claim 18
Dependent Claim
5 words
The plasmid pCVSVEHBVE400R400 or pETPAER400.
Claim 19
Dependent Claim
5 words
The plasmid pCVSVEHBVE400R400 or pETPAER400.
Claim 20
Independent Claim
28 words
A method of transforming eukaryotic cells which method comprises mixing said cells with the expression vector of any one of claims 3 to 6 and 8 to 12.
Claim 21
Independent Claim
46 words
A method of selecting transfected eukaryotic cells which method comprises mixing said cells with the expression vector of any one of claims 3 to 6 and 8 to 12 and growing the cells in a medium containing a concentration of MTX effective in selecting transformed cells.
Claim 22
Independent Claim
48 words
A method of amplifying the DNA sequence encoding a desired heterologous protein in a eukaryotic host cell culture, which method comprises mixing said cells with the vector of any one of claims 8 to 12 and growing the cells in the presence of am amplifying concentration of MTX.
Claim 23
Independent Claim
47 words
A method of increasing the production of a desired heterologous protein in a eukaryotic host cell culture, which method comprises mixing said cells with the vector of any one of claims 8 to 12 and growing the cells in the presence of an amplifying concentration of MTX.
Claim 24
Dependent Claim
17 words
A eukaryotic host cell culture transformed with the vector of any one of claims 8 to 12.
Claim 25
Independent Claim
19 words
The method of any one of claims 13, 15 and 16 wherein the host cell culture is CHO Kl.
Claim 26
Independent Claim
38 words
A method of selecting transfected vertebrate cells which method comprises providing a replicable expression vector comprising a DNA sequence encoding for a protein which confers drug resistance operably linked to a DNA sequence capable of effecting expression thereof.
Claim 27
Independent Claim
38 words
A method of selecting transfected vertebrate cells which method comprises providing a replicable expression vector comprising a DNA sequence encoding for a protein which confers drug resistance operably linked to a DNA sequence capable of effecting expression thereof.
Claim 28
Independent Claim
48 words
A method of selecting transfected vertebrate cells which method comprises providing a replicable expression vector comprising a DNA sequence encoding for desired heterologous protein and a DNA sequence encoding for a protein which confers drug resistance each operably linked to a DNA sequence capable of effecting expression thereof.
Claim 29
Independent Claim
48 words
A method of selecting transfected vertebrate cells which method comprises providing a replicable expression vector comprising a DNA sequence encoding for desired heterologous protein and a DNA sequence encoding for a protein which confers drug resistance each operably linked to a DNA sequence capable of effecting expression thereof.
Claim 30
Dependent Claim
16 words
A method according to claim 16 which further includes extracting the heterologous protein from the culture.
Claim 31
Dependent Claim
13 words
The method of claim 21 wherein the heterologous protein is HBsAg or tPA.
References Cited (Backward Citations)
No citation records indexed.
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