EP0336523A1
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Method for the in-vivo production and testing of proteins by recombinant gene expression in selected host-cells
Assignee: Intracel Corp
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Filing: 1989-04-06
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Granted: —
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Expires: 2009-04-06
Abstract
Abstract Eukaryotic host-cells transformed with stress-inducible gene constructs are inoculated into selected warm-blooded recipient animals multiplied either neat or as tumor fragments. Upon application of stress, either locally (tumor site) or as a whole, under harmless conditions, gene expression occurs and the gene products can be isolated from the body fluids of the animals. The response of the animals to the newly synthesized products can be observed directly, which eliminates all purification problems inherent to first isolating the substance before making physiological tests. Moreover the antibodies raised by the animal against the new product can be isolated by usual means or produced in-vitro from hybridoma strains involving cells taken from immunoorgans of the animals.
Inventors
- 👤 Michel Dreano
- 👤 Peter Bromley
- 👤 Richard Voellmy
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-29 20:29
CPC Classifications
A; A01; A01K; A01K67/00; A01K67/027; A01K67/0271; C; C07; C07K; C07K14/00; C07K14/435; C07K14/575; C07K14/61; C07K16/00; C07K16/18; C07K16/26; A01K2217/00; A01K2217/05
Claims Analysis
Total Claims: 20
Claim 1
Independent Claim
193 words
A method for directly producing in an animal one or more substances resulting from the expression in suitable host-cells of recombinant genes driven by a stress-inducible promotor, which comprises the steps of: a) selecting as host-cells suitable cell lines capable of being transfected by vectors carrying recombinant DNA and thereafter proliferating in vitro or in vivo, b) making a gene expression construct by placing a gene coding for said substance(s) under the expression control of stress inducible promotor element in a suitably engineered DNA vector and incorporating said vector into said host-cells, c) inoculating selected warm blooded recipient animals with said transformed host-cells said animals being capable to foster said inoculated cells and allow said recombinant gene to be expressed therein, d) rearing and nursing the animals for a period of time sufficient for them to develop a response consecutive to inoculation, e) allowing or causing the host cells in the animal to be subjected to stress under conditions sufficient to induce the in-vivo expression of said synthetic gene but insufficient to harm the animal, whereby said gene substance is expressed and becomes present in substantial amounts in the body of the animal.
Claim 2
Independent Claim
193 words
A method for directly producing in an animal one or more substances resulting from the expression in suitable host-cells of recombinant genes driven by a stress-inducible promotor, which comprises the steps of: a) selecting as host-cells suitable cell lines capable of being transfected by vectors carrying recombinant DNA and thereafter proliferating in vitro or in vivo, b) making a gene expression construct by placing a gene coding for said substance(s) under the expression control of stress inducible promotor element in a suitably engineered DNA vector and incorporating said vector into said host-cells, c) inoculating selected warm blooded recipient animals with said transformed host-cells said animals being capable to foster said inoculated cells and allow said recombinant gene to be expressed therein, d) rearing and nursing the animals for a period of time sufficient for them to develop a response consecutive to inoculation, e) allowing or causing the host cells in the animal to be subjected to stress under conditions sufficient to induce the in-vivo expression of said synthetic gene but insufficient to harm the animal, whereby said gene substance is expressed and becomes present in substantial amounts in the body of the animal.
Claim 3
Dependent Claim
38 words
The method of claim 1, in which the promotor is a heat-shock promotor and the gene product is a secretable protein which, in step e), is secreted in the blood stream or the ascite fluid of the animal.
Claim 4
Dependent Claim
21 words
The method of claim 1 or 2, in which the expressed gene product produces an observable physiological response in the animal.
Claim 5
Dependent Claim
20 words
The method of claim 1, in which the animals are either not immunodepressed, or they are naturally or chemically immunodepressed.
Claim 6
Dependent Claim
27 words
The method of claim 4, in which the animals are not immunodepressed, whereby stress occurs due to reactions of rejection by the animal in response to inoculation.
Claim 7
Dependent Claim
16 words
The method of claim 1, in which inoculation is carried out subcutaneously (sc) or intraperitoneally (ip).
Claim 8
Dependent Claim
18 words
The method of claim 1, in which the animals develop temporary or permanent tumors in response to inoculation.
Claim 9
Dependent Claim
20 words
The method of claim 7, in which stress involves heating the animal, either entirely, or locally at the tumoral site.
Claim 10
Dependent Claim
34 words
The method of claim 1, in which the animal's response includes raising antibodies against said substances expressed by said recombinant genes, said antibodies being available by extraction from the body fluids of the animals.
Claim 11
Dependent Claim
13 words
The method of claim 9, in which the antibody is isolated by immunoprecipitation.
Claim 12
Dependent Claim
25 words
The method of claim 1, in which the coding sequence of the gene to be expressed is cellular genomic DNA, or synthetic DNA, e.g. cDNA.
Claim 13
Dependent Claim
20 words
The method of claim 1, in which said gene expresses products selected from hormones, enzymes, viral antigens, growth factors, etc...
Claim 14
Dependent Claim
20 words
The method of claim 1, in which the recipient animals are selected from mice, rats, rabbits, hamsters and the like.
Claim 15
Dependent Claim
22 words
The method of claim 9, in which said antibodies are isolated from the blood stream or the ascite fluids of the animals.
Claim 16
Dependent Claim
31 words
The method of claim 6, in which said host cells to be inoculated are in the form of strains grown in vitro or of fragments of tumors grown in nude mice.
Claim 17
Dependent Claim
18 words
The method of claim 1, in which the animals are treated before being inoculated to develop aseptic peritonitis.
Claim 18
Dependent Claim
24 words
The method of claim 1, in which the host cells are either naturally tumorigenic, or they are made so by transfection with an oncogen.
Claim 19
Dependent Claim
24 words
The method of claim 17, in which said host cells are selected from mouse fibroblasts, human amniotic cells, hamster ovary cells and the like.
Claim 20
Dependent Claim
37 words
The method of claim 4, in which the antibodies producing cells are removed from the animals, fused with strains of carcinoma cells to provide hybridoma cells and in which these cells are multiplied to produce monoclonal antibodies.
References Cited (Backward Citations)
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