US4663281A
Expired / Lapsed
US
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Enhanced production of proteinaceous materials in eucaryotic cells
Assignee: Massachusetts Institute of Technology
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Filing: 1984-03-22
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Granted: 1987-05-05
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Expires: 2004-03-22
Abstract
Abstract Disclosed is a method of enhancing expression of recombinant DNA in eucaryotic cells. A tissue specific enhancer element obtained from the genome of an organism and active in a selected tissue type is combined with a transcriptionally competent transcription unit comprising a promoter and exons encoding for the proteinaceous material of interest (or its precursor). This recombinant DNA is transfected into cells derived from the same tissue as the tissue in which the enhancer element normally functions to enhance expression of endogeneous DNA. The resulting transformants express the exons of the transcription unit at high levels as the enhancer element increases the copy number of mRNA. The enhancer element operates to increase transcription independent of its orientation and position provided it is located within an active region on the DNA, generally between about 1-10 kilobases (kb) from the 3' or 5' end of the transcription unit.
Inventors
- 👤 Stephen D. Gillies
- 👤 Susumu Tonegawa
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 16:03
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/67; C07; C07K; C07K16/00; C12N15/79; C12N15/85
Claims Analysis
Total Claims: 29
Claim 1
Independent Claim
110 words
A process for producing a proteinaceous material in a mammalian cell line derived from a selected tissue type comprising the steps of: combining DNA comprising a mammalian a tissue specific cellular enhancer element with DNA comprising a transcription unit encoding said proteinaceous material or a precursor thereof to produce transcriptionally competent recombinant DNA, said tissue specific cellular enhancer element, when present in the endogenous genome of a cell from said selected tissue type, being operable naturally to increase the production of an endogenous proteinaceous substance; transfecting cells of said mammalian cell line with said recombinant DNA; and culturing said transfected cell line to produce enhanced quantities of said proteinaceous material.
Claim 2
Dependent Claim
110 words
A process for producing a proteinaceous material in a mammalian cell line derived from a selected tissue type comprising the steps of: combining DNA comprising a mammalian a tissue specific cellular enhancer element with DNA comprising a transcription unit encoding said proteinaceous material or a precursor thereof to produce transcriptionally competent recombinant DNA, said tissue specific cellular enhancer element, when present in the endogenous genome of a cell from said selected tissue type, being operable naturally to increase the production of an endogenous proteinaceous substance; transfecting cells of said mammalian cell line with said recombinant DNA; and culturing said transfected cell line to produce enhanced quantities of said proteinaceous material.
Claim 3
Dependent Claim
42 words
The process of claim 1 wherein said enhancer element, when present in the endogenous genome of a cell from said selected tissue type, functions to enhance production of a proteinaceous substance selected from the group consisting of albumin, globulins, fibrinogen, and hormones.
Claim 4
Dependent Claim
26 words
The process of claim 1 wherein said combining step includes combining said recombinant DNA with a vector selected from the group consisting of plasmids and viruses.
Claim 5
Dependent Claim
22 words
The process of claim 3 wherein said vector further includes expressable DNA which corresponds to a gene coding for a selectable marker.
Claim 6
Dependent Claim
18 words
The process of claim 1 wherein said transfecting and culturing steps result in a stably transformed cell line.
Claim 7
Dependent Claim
28 words
The process of claim 1 wherein said enhancer element, when present in the endogenous genome of a lymphoid cell, functions to enhance expression of DNA encoding a globulin.
Claim 8
Dependent Claim
32 words
The process of claim 1 wherein said enhancer element comprises at least a portion of the nucleotide sequences set forth in FIG. 10 and said cell line is a lymphoid cell line.
Claim 9
Dependent Claim
34 words
The process of claim 1 wherein said cell line is a myeloma cell line and said enhancer element, when present in the endogenous genome of a lymphoid cell, enhances the production of an immunoglobulin.
Claim 10
Dependent Claim
31 words
The process of claim 1 wherein the cell line transfected with said recombinant DNA is derived from the same cell type as the cell in which said enhancer element is active.
Claim 11
Dependent Claim
111 words
A vector for transfecting a mammalian cell derived from a selected tissue type to produce a cell line which secretes a proteinaceous material, said vector comprising an exon encoding said proteinaceous material or a precursor thereof and a promoter sequence, and recombined therewith, tissue specific mammalian cellular enhancer element at a site within an active region of said vector sufficiently close to said transcription unit to enhance production of mRNA independent of its orientation and position within said active region, said tissue specific cellular enhancer element, when present in the endogenous genome of a cell from said selected tissue type, being operable naturally to increase production of an endogenous proteinaceous substance.
Claim 12
Dependent Claim
111 words
A vector for transfecting a mammalian cell derived from a selected tissue type to produce a cell line which secretes a proteinaceous material, said vector comprising an exon encoding said proteinaceous material or a precursor thereof and a promoter sequence, and recombined therewith, tissue specific mammalian cellular enhancer element at a site within an active region of said vector sufficiently close to said transcription unit to enhance production of mRNA independent of its orientation and position within said active region, said tissue specific cellular enhancer element, when present in the endogenous genome of a cell from said selected tissue type, being operable naturally to increase production of an endogenous proteinaceous substance.
Claim 13
Dependent Claim
43 words
The vector of claim 10 wherein said enhancer element, when present in the endogenous genome of a cell from said selected tissue type, functions to enhance the production of a proteinaceous substance selected from the group consisting of albumin, globulins, fibrinogen, and hormones.
Claim 14
Dependent Claim
19 words
The vector of claim 10 further comprising expressable DNA which corresponds to a gene coding for a selectable marker.
Claim 15
Dependent Claim
17 words
The vector of claim 10 comprising a member selected from the group consisting of plasmids and viruses.
Claim 16
Dependent Claim
19 words
The vector of claim 10 wherein said enhancer element and said transcription unit are derived from different cell lines.
Claim 17
Dependent Claim
27 words
The vector of claim 10 wherein said enhancer element, when present in the endogenous genome of a lymphoid cell, is operative naturally to enhance production of immunoglobulin.
Claim 18
Dependent Claim
40 words
The vector of claim 10 wherein said enhancer element, when present as part of the endogenous genome of a lymophoid cell in an intron of a γ2b heavy chain gene, serves to enhance production of a γ2b heavy chain protein.
Claim 19
Dependent Claim
23 words
The vector of claim 10 wherein said enhancer element comprises at least a portion of the nucleotide sequences set forth in FIG. 10.
Claim 20
Dependent Claim
117 words
A mammalian cell transformant for producing a proteinaceous material, said transformant comprising a genetically modified cell derived from a selected mammalian tissue type containing a transfected DNA comprising: a transcription unit comprising an exon encoding said proteinaceous material or a precursor thereof and a promoter sequence; and, recombined therewith, tissue specific a mammalian cellular enhancer element at a site within an active region of said DNA sufficiently close to said transcription unit to enhance production of mRNA independent of orientation and position within said active region, said tissue specific cellular enhancer element, when present in the endogenous genome of a cell from said selected tissue type, being operative naturally to enhance production of an endogenous proteinaceous substance.
Claim 21
Dependent Claim
117 words
A mammalian cell transformant for producing a proteinaceous material, said transformant comprising a genetically modified cell derived from a selected mammalian tissue type containing a transfected DNA comprising: a transcription unit comprising an exon encoding said proteinaceous material or a precursor thereof and a promoter sequence; and, recombined therewith, tissue specific a mammalian cellular enhancer element at a site within an active region of said DNA sufficiently close to said transcription unit to enhance production of mRNA independent of orientation and position within said active region, said tissue specific cellular enhancer element, when present in the endogenous genome of a cell from said selected tissue type, being operative naturally to enhance production of an endogenous proteinaceous substance.
Claim 22
Dependent Claim
43 words
The transformant of claim 18 wherein said enhancer element, when present in the endogenous genome of a cell from said selected tissue type, functions to enhance the production of a proteinaceous substance selected from the group consisting of albumin, globulins, fibrinogen, and hormones.
Claim 23
Dependent Claim
34 words
The transformant of claim 18 wherein said genetically modified cell is a lymphoid cell and said enhancer element, when present in the endogenous genome of a lymphoid cell, enhances the production of an immunoglobulin.
Claim 24
Dependent Claim
10 words
The transformant of claim 18 comprising a stably transformed cell.
Claim 25
Dependent Claim
23 words
The transformant of claim 20 wherein said enhancer element comprises at least a portion of the nucleotide sequences set forth in FIG. 10.
Claim 26
Dependent Claim
10 words
The transformant of claim 18 comprising a genetically modified cell.
Claim 27
Dependent Claim
112 words
A vector for receiving DNA encoding a proteinaceous material and for transfecting a mammalian cell derived from a selected tissue type to produce a cell line which produces said proteinaceous material, said vector comprising a restriction site for receiving a gene encoding said proteinaceous material and tissue specific a mammalian cellular enhancer element located within an active region of said vector sufficiently close to said restriction site to enhance production of mRNA corresponding to a gene inserted into said restriction site, said tissue specific cellular enhancer element, when present in the endogenous genome of a cell from said selected tissue type, being operable naturally to increase production of an endogenous proteinaceous substance.
Claim 28
Dependent Claim
112 words
A vector for receiving DNA encoding a proteinaceous material and for transfecting a mammalian cell derived from a selected tissue type to produce a cell line which produces said proteinaceous material, said vector comprising a restriction site for receiving a gene encoding said proteinaceous material and tissue specific a mammalian cellular enhancer element located within an active region of said vector sufficiently close to said restriction site to enhance production of mRNA corresponding to a gene inserted into said restriction site, said tissue specific cellular enhancer element, when present in the endogenous genome of a cell from said selected tissue type, being operable naturally to increase production of an endogenous proteinaceous substance.
Claim 29
Dependent Claim
14 words
The vector of claim 27 further comprising a promoter upstream of said restriction site.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1990-05-04 | 1990-11-03 | 1991-05-05 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1994-05-04 | 1994-11-03 | 1995-05-05 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1998-05-04 | 1998-11-03 | 1999-05-05 | $7700.00 | Window Closed / Lapsed |