US4399216A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
Assignee: Columbia University
•
Filing: 1980-02-25
•
Granted: 1983-08-16
•
Expires: 2000-02-25
Abstract
Abstract The present invention relates to processes for inserting DNA into eucaryotic cells, particularly DNA which includes a gene or genes coding for desired proteinaceous materials for which no selective criteria exist. The insertion of such DNA molecules is accomplished by cotransforming eucaryotic cells with such DNA together with a second DNA which corresponds to a gene coding for a selectable marker. The invention further relates to processes for inserting into eucaryotic cells a multiplicity of DNA molecules including genes coding for desired proteinaceous materials by cotransformation with the desired genes and with amplifiable genes for a dominant selectable marker in the presence of successively higher amounts of an inhibitor. Alternatively, the insertion of multiple copies of desired genes is accomplished by transformation using DNA molecules formed by ligating a DNA molecule including the desired gene to a DNA molecule which includes an amplifiable gene coding for a dominant selectable phenotype such as a gene associated with resistance to a drug in the presence of successively higher amounts of an agent such as a drug against which the gene confers resistance so that only those eucaryotic cells into which multiple copies of the amplifiable gene have been inserted survive.
Inventors
- 👤 Richard Axel
- 👤 Michael H. Wigler
- 👤 Saul J. Silverstein
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 04:27
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/87; C12N15/63; C12N15/79; C12N15/85; C12P; C12P21/00; C12P21/02; Y; Y10; Y10S; Y10S435/00; Y10S435/811; Y10S435/8215; Y10S435/948
Claims Analysis
Total Claims: 79
Claim 1
Independent Claim
76 words
A process for inserting foreign DNA I into a suitable eucaryotic cell which comprises cotransforming said eucaryotic cell with said foreign DNA I and with unlinked foreign DNA II which codes for a selectable phenotype not expressed by said ducaryotic cell, said cotransformation being carried out under suitable conditions permitting survival or identification of eucaryotic cells which have acquired said selectable phenotype, said foreign DNA I being incorporated into the chromosomal DNA of said eucaryotic cell.
Claim 2
Dependent Claim
76 words
A process for inserting foreign DNA I into a suitable eucaryotic cell which comprises cotransforming said eucaryotic cell with said foreign DNA I and with unlinked foreign DNA II which codes for a selectable phenotype not expressed by said ducaryotic cell, said cotransformation being carried out under suitable conditions permitting survival or identification of eucaryotic cells which have acquired said selectable phenotype, said foreign DNA I being incorporated into the chromosomal DNA of said eucaryotic cell.
Claim 3
Dependent Claim
24 words
A process in accordance with claim 1 wherein said foreign DNA I codes for proteinaceous material which is not associated with a selectable phenotype.
Claim 4
Dependent Claim
16 words
A process in accordance with claim 2 wherein said foreign DNA I codes for interferon protein.
Claim 5
Dependent Claim
15 words
A process in accordance with claim 2 wherein said foreign DNA I codes for insulin.
Claim 6
Dependent Claim
16 words
A process in accordance with claim 2 wherein said foreign DNA I codes for growth hormone.
Claim 7
Dependent Claim
17 words
A process in accordance with claim 2 wherein said foreign DNA I codes for a clotting factor.
Claim 8
Dependent Claim
20 words
A process in accordance with claim 2 wherein said foreign DNA I codes for a viral antigen or an antibody.
Claim 9
Dependent Claim
16 words
A process in accordance with claim 2 wherein said foreign DNA I codes for an enzyme.
Claim 10
Dependent Claim
15 words
A process in accordance with claim 1 wherein said foreign DNA I is substantially purified.
Claim 11
Dependent Claim
23 words
A process in accordance with claim 1 wherein said foreign DNA I has been obtained from restriction endonuclease cleavage of eucaryotic chromosomal DNA.
Claim 12
Dependent Claim
21 words
A process in accordance with claim 1 wherein said foreign DNA I and DNA II have been treated with calcium phosphate.
Claim 13
Dependent Claim
15 words
A process in accordance with claim 1 wherein said eucaryotic cell is a mammalian cell.
Claim 14
Dependent Claim
14 words
A process in accordance with claim 12 wherein said mammalian cell is an erythroblast.
Claim 15
Dependent Claim
14 words
A process in accordance with claim 12 wherein said mammalian cell is a fibroblast.
Claim 16
Dependent Claim
37 words
A process in accordance with claim 1 wherein said foreign DNA I is present in an amount relative to said DNA II which codes for a selectable phenotype in the range from about 1:1 to about 100,000:1.
Claim 17
Dependent Claim
27 words
A process in accordance with claim 1 wherein said DNA II which codes for a selectable phenotype comprises the gene for thymidine kinase from herpes simplex virus.
Claim 18
Dependent Claim
29 words
A process in accordance with claim 1 wherein said DNA II which codes for proteinaceous material which is associated with a selectable phenotype comprises the gene for adenine phosphoribosyltransferase.
Claim 19
Dependent Claim
24 words
A process in accordance with claim 1 wherein said DNA II which codes for a selectable phenotype comprises a gene associated with drug resistance.
Claim 20
Dependent Claim
29 words
A process in accordance with claim 18 wherein said gene associated with drug resistance is the gene coding for a mutant dihydrofolate reductase which renders cells resistant to methotrexate.
Claim 21
Dependent Claim
19 words
A eucaryotic cell into which foreign DNA I has been inserted in accordance with the process of claim 1.
Claim 22
Dependent Claim
19 words
A mammalian cell into which foreign DNA I has been inserted in accordance with the process of claim 1.
Claim 23
Dependent Claim
53 words
A process for producing a foreign proteinaceous material which comprises cotransforming a eucaryotic cell in accordance with the process of claim 1, culturing or cloning said cotransformed eucaryotic cell under suitable conditions to yield a multiplicity of eucaryotic cells producing said foreign proteninaceous material and recovering said proteinaceous material from said eucaryotic cells.
Claim 24
Dependent Claim
23 words
A process in accordance with claim 22 wherein said proteinaceous material comprises interferon protein, insulin, growth hormone, clotting factor, viral antigen or antibody.
Claim 25
Dependent Claim
15 words
A process in accordance with claim 22 wherein said eucaryotic cell is a mammalian cell.
Claim 26
Dependent Claim
58 words
A method of detecting eucaryotic cells which have been transformed with foreign DNA I which is not associated with a selectable phenotype which comprises cotransforming said eucaryotic cell with said DNA I and with DNA II which is associated with a selectable phenotype in accordance with the process of claim 1, and screening for eucaryotic cells so cotransformed.
Claim 27
Dependent Claim
68 words
A process for inserting foreign DNA I into a eucaryotic cell which comprises cotransforming said eucaryotic cell with said foreign DNA I and with unlinked foreign DNA II which codes for a selectable phenotype not expressed by said eucaryotic cell, said cotransformation being carried out in a suitable medium and in the presence of conditions permitting identification and recovery of eucaryotic cells which have acquired said selectable phenotype.
Claim 28
Dependent Claim
68 words
A process for inserting foreign DNA I into a eucaryotic cell which comprises cotransforming said eucaryotic cell with said foreign DNA I and with unlinked foreign DNA II which codes for a selectable phenotype not expressed by said eucaryotic cell, said cotransformation being carried out in a suitable medium and in the presence of conditions permitting identification and recovery of eucaryotic cells which have acquired said selectable phenotype.
Claim 29
Dependent Claim
52 words
A process for cotransforming a suitable eucaryotic cell which comprises transforming under suitable conditions said eucaryotic cell with foreign DNA I and with foreign DNA II, said DNA I and DNA II being unlinked and said DNA II coding for a selectable phenotype not expressed by said eucaryotic cell prior to cotransformation.
Claim 30
Dependent Claim
52 words
A process for cotransforming a suitable eucaryotic cell which comprises transforming under suitable conditions said eucaryotic cell with foreign DNA I and with foreign DNA II, said DNA I and DNA II being unlinked and said DNA II coding for a selectable phenotype not expressed by said eucaryotic cell prior to cotransformation.
Claim 31
Dependent Claim
88 words
A process for inserting purified foreign DNA I coding for proteinaceous material which is not associated with a selectable phenotype into a suitable eucaryotic cell which comprises cotransforming said eucaryotic cell with said foreign DNA I and with unlinked foreign DNA II coding for proteinaceous material which is associated with a selectable phenotype, said cotransformation being carried out under suitable conditions permitting survival or identification of eucaryotic cells which have acquired said selectable phenotype, said foreign DNA I being incorporated into the chromosomal DNA of said eucaryotic cell.
Claim 32
Dependent Claim
88 words
A process for inserting purified foreign DNA I coding for proteinaceous material which is not associated with a selectable phenotype into a suitable eucaryotic cell which comprises cotransforming said eucaryotic cell with said foreign DNA I and with unlinked foreign DNA II coding for proteinaceous material which is associated with a selectable phenotype, said cotransformation being carried out under suitable conditions permitting survival or identification of eucaryotic cells which have acquired said selectable phenotype, said foreign DNA I being incorporated into the chromosomal DNA of said eucaryotic cell.
Claim 33
Dependent Claim
31 words
A process in accordance with claim 28 wherein said proteinaceous material which is not associated with a selectable phenotype comprises interferon protein, insulin, growth hormone, clotting factor, viral antigen or antibody.
Claim 34
Dependent Claim
19 words
A eucaryotic cell into which foreign DNA I has been inserted in accordance with the process of claim 28.
Claim 35
Dependent Claim
90 words
A process for inserting a multiplicity of foreign DNA I molecules corresponding to multiple copies of a gene coding for a proteinaceous material into a suitable eucaryotic cell which comprises cotransforming said eucaryotic cell with said multiplicity of foreign DNA I molecules and with a multiplicity of unlinked foreign DNA II molecules coding for a selectable phenotype not expressed by said eucaryotic cell, said cotransformation being carried out under suitable conditions permitting survival or identification of eucaryotic cells which have acquired said multiplicity of genes coding for said selectable phenotype.
Claim 36
Dependent Claim
90 words
A process for inserting a multiplicity of foreign DNA I molecules corresponding to multiple copies of a gene coding for a proteinaceous material into a suitable eucaryotic cell which comprises cotransforming said eucaryotic cell with said multiplicity of foreign DNA I molecules and with a multiplicity of unlinked foreign DNA II molecules coding for a selectable phenotype not expressed by said eucaryotic cell, said cotransformation being carried out under suitable conditions permitting survival or identification of eucaryotic cells which have acquired said multiplicity of genes coding for said selectable phenotype.
Claim 37
Dependent Claim
24 words
A process in accordance with claim 31 wherein said foreign DNA I codes for proteinaceous material which is not associated with a selectable phenotype.
Claim 38
Dependent Claim
16 words
A process in accordance with claim 32 wherein said foreign DNA I codes for interferon protein.
Claim 39
Dependent Claim
15 words
A process in accordance with claim 32 wherein said foreign DNA I codes for insulin.
Claim 40
Dependent Claim
16 words
A process in accordance with claim 32 wherein said foreign DNA I codes for growth hormone.
Claim 41
Dependent Claim
17 words
A process in accordance with claim 32 wherein said foreign DNA I codes for a clotting factor.
Claim 42
Dependent Claim
20 words
A process in accordance with claim 32 wherein said foreign DNA I codes for a viral antigen or an antibody.
Claim 43
Dependent Claim
16 words
A process in accordance with claim 32 wherein said foreign DNA I codes for an enzyme.
Claim 44
Dependent Claim
15 words
A process in accordance with claim 31 wherein said foreign DNA I is substantially purified.
Claim 45
Dependent Claim
23 words
A process in accordance with claim 31 wherein said foreign DNA I has been obtained from restriction endonuclease cleavage of eucaryotic chromosomal DNA.
Claim 46
Dependent Claim
21 words
A process in accordance with claim 31 wherein said foreign DNA I and DNA II have been treated with calcium phosphate.
Claim 47
Dependent Claim
15 words
A process in accordance with claim 31 wherein said eucaryotic cell is a mammalian cell.
Claim 48
Dependent Claim
14 words
A process in accordance with claim 42 wherein said mammalian cell is an erythroblast.
Claim 49
Dependent Claim
14 words
A process in accordance with claim 42 wherein said mammalian cell is a fibroblast.
Claim 50
Dependent Claim
41 words
A process in accordance with claim 31 wherein said foreign DNA I is present in an amount relative to said DNA II which codes for proteinaceous material associated with a selectable phenotype in the range from about 1:1 to about 100,000:1.
Claim 51
Dependent Claim
31 words
A process in accordance with claim 31 wherein said foreign DNA II which does for proteinaceous material which is associated with a selectable phenotype comprises a gene associated with drug resistance.
Claim 52
Dependent Claim
29 words
A process in accordance with claim 46 wherein said gene associated with drug resistance is a gene coding for a mutant dihydrofolate reductase which renders cells resistant to methotrexate.
Claim 53
Dependent Claim
22 words
A process in accordance with claim 31 wherein said foreign DNA I is incorporated into the chromosomal DNA of said eucaryotic cell.
Claim 54
Dependent Claim
19 words
A eucaryotic cell into which foreign DNA I has been inserted in accordance with the process of claim 31.
Claim 55
Dependent Claim
19 words
A mammalian cell into which foreign DNA I has been inserted in accordance with the process of claim 31.
Claim 56
Dependent Claim
43 words
A process for producing a foregin proteinaceous material which comprises cotransforming a eucaryotic cell in accordance with the process of claim 31, maintaining said cotransformed eucaryotic cell under suitable conditions to produce said foreign proteinaceous material, and recovering said proteinaceous material so produced.
Claim 57
Dependent Claim
23 words
A process in accordance with claim 51 wherein said proteinaceous material comprises interferon protein, insulin, growth hormone, clotting factor, viral antigen or antibody.
Claim 58
Dependent Claim
14 words
A process in accordance with claim 51 wherein said eucaryotic cell is mammalian cell.
Claim 59
Dependent Claim
106 words
A process for generating a multiplicity of foregin DNA I molecules corresponding to multiple copies of a gene in a eucaryotic cell which comprises tranforming said eucaryotic cell with a molecule which is formed by linking one of said foreign DNA I molecules to a DNA II molecule corresponding to an amplifiable gene for a dominant selectable phenotype not expressed by said eucaryotic cell, and culturing the transformed eucaryotic cells in the presence of successively elevated concentrations of an agent permitting survival or identification of eucaryotic cells which have acquired multiple copies of said amplifiable gene, said transformation and culturing being carried out under suitable conditions.
Claim 60
Dependent Claim
106 words
A process for generating a multiplicity of foregin DNA I molecules corresponding to multiple copies of a gene in a eucaryotic cell which comprises tranforming said eucaryotic cell with a molecule which is formed by linking one of said foreign DNA I molecules to a DNA II molecule corresponding to an amplifiable gene for a dominant selectable phenotype not expressed by said eucaryotic cell, and culturing the transformed eucaryotic cells in the presence of successively elevated concentrations of an agent permitting survival or identification of eucaryotic cells which have acquired multiple copies of said amplifiable gene, said transformation and culturing being carried out under suitable conditions.
Claim 61
Dependent Claim
24 words
A process in accordance with claim 54 wherein said foreign DNA I codes for proteinaceous material which is not associated with a selectable phenotype.
Claim 62
Dependent Claim
16 words
A process in accordance with claim 55 wherein said foreign DNA I codes for interferon protein.
Claim 63
Dependent Claim
15 words
A process in accordance with claim 55 wherein said foreign DNA I codes for insulin.
Claim 64
Dependent Claim
16 words
A process in accordance with claim 55 wherein said foreign DNA I codes for growth hormone.
Claim 65
Dependent Claim
17 words
A process in accordance with claim 55 wherein said foreign DNA I codes for a clotting factor.
Claim 66
Dependent Claim
19 words
A process in accordance with claim 55 wherein said foreign DNA I codes for a viral antigen or antibody.
Claim 67
Dependent Claim
16 words
A process in accordance with claim 55 wherein said foregin DNA I codes for an enzyme.
Claim 68
Dependent Claim
15 words
A process in accordance with claim 54 wherein said foreign DNA I is substantially purified.
Claim 69
Dependent Claim
23 words
A process in accordance with claim 54 wherein said foreign DNA I has been obtained from restriction endonuclease cleavage of eucaryotic chromosomal DNA.
Claim 70
Dependent Claim
21 words
A process in accordance with claim 54 wherein said foreign DNA I and DNA II have been treated with calcium phosphate.
Claim 71
Dependent Claim
14 words
A process in accordance with claim 54 wherein said eucaryotic cell is mammalian cell.
Claim 72
Dependent Claim
14 words
A process in accordance with claim 65 wherein said mammalian cell is an erythroblast.
Claim 73
Dependent Claim
14 words
A process in accordance with claim 65 wherein said mammalian cell is a fibroblast.
Claim 74
Dependent Claim
41 words
A process in accordance with claim 54 wherein said foreign DNA I is present in an amount relative to said DNA II which codes for proteinaceous material associated with a selectable phenotype in the range from about 1:1 to about 100,000:1.
Claim 75
Dependent Claim
35 words
A process in accordance with claim 54 wherein said DNA II which codes for proteinaceous material which is associated with a selectable phenotype comprises a gene associated with resistance to a drug or chemical antagonist.
Claim 76
Dependent Claim
34 words
A process in accordance with claim 69 wherein said gene associated with resistance to a drug or chemical anatgonist is a gene coding for a mutant dihydrofolate reductase which renders cells resistant to methotrexate.
Claim 77
Dependent Claim
22 words
A process in accordance with claim 54 wherein said foreign DNA I is incorporated into the chromosomal DNA of said eucaryotic cell.
Claim 78
Dependent Claim
19 words
A eucaryotic cell into which foreign DNA I has been inserted in accordance with the process of claim 54.
Claim 79
Dependent Claim
19 words
A mammalian cell into which foreign DNA I has been inserted in accordance with the process of claim 54.
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) | 1986-08-15 | 1987-02-14 | 1987-08-16 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1990-08-15 | 1991-02-14 | 1991-08-16 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1994-08-15 | 1995-02-14 | 1995-08-16 | $7700.00 | Window Closed / Lapsed |