US5270191A
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Method for manipulation of the cell types of eukaryotes
Assignee: Massachusetts Institute of Technology
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Filing: 1992-05-12
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Granted: 1993-12-14
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Expires: 2012-05-12
Abstract
Abstract A novel method of immortalizing cell lines, as well as cell lines immortalized by the method. According to the method of the present invention, a gene which confers on a cell the ability to grow in tissue culture is introduced, using known techniques, into such a cell. This gene, referred to as a growth promoting gene, is under control such that when the gene action or function is turned down or off, the cell in which it resides switches to a new differentiated state. In one embodiment, the temperature sensitive (ts) form of the oncogene derived from the simian virus SV40 is the growth-promoting gene.
Inventors
- 👤 Ronald D. G. McKay
- 👤 Parmjit S. Jat
- 👤 Guillermina Almazan
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-03 09:34
CPC Classifications
C; C12; C12N; C12N5/00; C12N5/06; C12N5/0602; C12N5/0618; C12N5/0623; C12N2510/00; C12N2510/04
Claims Analysis
Total Claims: 14
Claim 1
Independent Claim
163 words
A method of immortalizing mammalian neural cells, to produce immortalized cells which are capable of multipotential differentiation, comprising the steps of: a) growing embryonic mammalian neural precursor cells in primary culture under permissive conditions; b) selecting embryonic mammalian neural precursor cells which are capable of multipotential differentiation with an antibody which recognizes a marker protein expressed by neural precursor cells which is recognized by Rat 401 antibody; d) introducing into appropriately-selected mammalian neural precursor cells a growth-promoting gene which is the temperature sensitive domain of the tsA58 strain of the Sv40 virus and is activated under said permissive conditions, said growth-promoting gene conferring on said neural precursor cells the ability to grow in tissue culture, to produce immortalized cells containing said growth-promoting gene which are capable of multipotential differentiation; and d) selecting neural cells containing said growth-promoting gene which are capable of multipotential differentiation and maintaining them under permissive conditions and in medium appropriate for cell growth, for sufficient time for cell growth.
Claim 2
Independent Claim
163 words
A method of immortalizing mammalian neural cells, to produce immortalized cells which are capable of multipotential differentiation, comprising the steps of: a) growing embryonic mammalian neural precursor cells in primary culture under permissive conditions; b) selecting embryonic mammalian neural precursor cells which are capable of multipotential differentiation with an antibody which recognizes a marker protein expressed by neural precursor cells which is recognized by Rat 401 antibody; d) introducing into appropriately-selected mammalian neural precursor cells a growth-promoting gene which is the temperature sensitive domain of the tsA58 strain of the Sv40 virus and is activated under said permissive conditions, said growth-promoting gene conferring on said neural precursor cells the ability to grow in tissue culture, to produce immortalized cells containing said growth-promoting gene which are capable of multipotential differentiation; and d) selecting neural cells containing said growth-promoting gene which are capable of multipotential differentiation and maintaining them under permissive conditions and in medium appropriate for cell growth, for sufficient time for cell growth.
Claim 3
Dependent Claim
34 words
Immortalized embryonic mammalian cells containing a growth promoting gene which is the temperature-sensitive domain of the tsA58 strain of the SV40 virus and capable of multipotential differentiation produced by the method of claim 1.
Claim 4
Independent Claim
118 words
A method of producing, differentiated mammalian neural cells, comprising; a) introducing into embryonic mammalian neural precursor cells, which have been selectively isolated with an antibody which recognizes a marker protein expressed by neural precursor cells which is recognized by Rat 401 antibody and are capable of multipotential differentiation, a retroviral vector comprising a growth-promoting gene which is the temperature-sensitive domain of the tsA58 strain of the SV40 virus; b) culturing cells produced in (a) at a temperature of approximately 33° C. and under conditions sufficient for cell growth; c) shifting the temperature of the cultured cells to approximately 39° C.; and d) maintaining the cells under conditions appropriate for and for sufficient time for cell differentiation to occur.
Claim 5
Independent Claim
118 words
A method of producing, differentiated mammalian neural cells, comprising; a) introducing into embryonic mammalian neural precursor cells, which have been selectively isolated with an antibody which recognizes a marker protein expressed by neural precursor cells which is recognized by Rat 401 antibody and are capable of multipotential differentiation, a retroviral vector comprising a growth-promoting gene which is the temperature-sensitive domain of the tsA58 strain of the SV40 virus; b) culturing cells produced in (a) at a temperature of approximately 33° C. and under conditions sufficient for cell growth; c) shifting the temperature of the cultured cells to approximately 39° C.; and d) maintaining the cells under conditions appropriate for and for sufficient time for cell differentiation to occur.
Claim 6
Dependent Claim
28 words
Differentiated mammalian neural cells, containing a growth-promoting gene which is the temperature-sensitive domain of the tsA58 strain of the SV40 virus, produced by the method of claim 3.
Claim 7
Dependent Claim
16 words
Differentiated cells of claim 4 selected from the group consisting of neuronal cells and glial cells.
Claim 8
Dependent Claim
97 words
An immortalized cell line, capable of multipotential differentiation produced by incorporating into embryonic mammalian nervous system cells, which have been selectively isolated with an antibody which recognizes a marker protein expressed by neural precursor cells which are recognized by Rat 401 antibody and are capable of multipotential differentiation, a retroviral vector comprising a growth-promoting gene which is the temperature-sensitive domain of the tsA58 strain of the Sv40 virus and a gene encoding drug resistance, culturing said cells at a temperature of approximately 33° C. under conditions sufficient for and for sufficient time for cell growth to occur.
Claim 9
Dependent Claim
97 words
An immortalized cell line, capable of multipotential differentiation produced by incorporating into embryonic mammalian nervous system cells, which have been selectively isolated with an antibody which recognizes a marker protein expressed by neural precursor cells which are recognized by Rat 401 antibody and are capable of multipotential differentiation, a retroviral vector comprising a growth-promoting gene which is the temperature-sensitive domain of the tsA58 strain of the Sv40 virus and a gene encoding drug resistance, culturing said cells at a temperature of approximately 33° C. under conditions sufficient for and for sufficient time for cell growth to occur.
Claim 10
Dependent Claim
26 words
The method of immortalizing mammalian neural cells of claim 1 wherein in step b, the embryonic mammalian neural precursor cells are selected by Rat 401 antibody.
Claim 11
Dependent Claim
28 words
The method of producing differentiated mammalian neural cells of claim 3, wherein in step a the embryonic mammalian neural precursor cells are selectively isolated using Rat 401 antibody.
Claim 12
Dependent Claim
21 words
The immortalized cell line of claim 6 wherein the embryonic mammalian nervous system cells are selectively isolated by Rat 401 antibody.
Claim 13
Dependent Claim
38 words
The oligodendrocyte optic nerve cell line, ts U195, containing a growth-promoting gene which is the temperature sensitive domain of the tsA58 stain of the SV40 virus, deposited at the American Type Culture Collection under accession number CRL 9729.
Claim 14
Dependent Claim
38 words
The oligodendrocyte optic nerve cell line, ts U195, containing a growth-promoting gene which is the temperature sensitive domain of the tsA58 stain of the SV40 virus, deposited at the American Type Culture Collection under accession number CRL 9729.
References Cited (Backward Citations)
US4707448A
Cited
WO1989003872A1
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1996-12-13 | 1997-06-14 | 1997-12-14 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2000-12-13 | 2001-06-14 | 2001-12-14 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2004-12-13 | 2005-06-14 | 2005-12-14 | $7700.00 | Window Closed / Lapsed |