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TRANSGENIC NON-HUMAN MAMMAL WITH AN ONCOGENIC MUTANT OF THE c-Raf GENE 1

a technology of c-raf and non-human mammal, which is applied in the field of transgenic non-human mammals, can solve the problems of little success of therapy predictions, and achieve the effects of reducing alveolar surface tension, high selectivity and reproducibility

Inactive Publication Date: 2006-09-21
RAPP ULF
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The invention is about creating non-human mammals that have a high number of tumors for pre-clinic examinations of anti-cancer substances and therapies. The invention achieves this by introducing a foreign DNA that contains a constitutively active oncogenic mutant of the kinase-domain of the c-Raf-1 gene or a corresponding normal allele or derivative of the gene. The foreign DNA can also contain DNA of the SV40 virus, which increases the translation efficiency of the polyadenylated mRNA. The invention provides a way to obtain a large number of mammals with reproducible tumors for pre-clinic examinations with statistical significance."

Problems solved by technology

Cancer, in particular lung cancer, is one of the most widespread diseases of mankind, and has up to now therapy predictions offering little success only.

Method used

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  • TRANSGENIC NON-HUMAN MAMMAL WITH AN ONCOGENIC MUTANT OF THE c-Raf GENE 1
  • TRANSGENIC NON-HUMAN MAMMAL WITH AN ONCOGENIC MUTANT OF THE c-Raf GENE 1
  • TRANSGENIC NON-HUMAN MAMMAL WITH AN ONCOGENIC MUTANT OF THE c-Raf GENE 1

Examples

Experimental program
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Effect test

example 1

[0029] Cloning of the cDNA sequences of the transforming c-Raf subdomain into a lung-specific expression vector was performed as follows. The vector SPC-Raf-1 for generating the wild type-Raf-1 transgenic mice was produced by that a 3. 0 kb fragment of the human Raf-1 cDNA (Bonner, T. I.; Oppermann, R.; Seebrug, P.; Kerby, S. B,; Gunnell, M. A.; Young, A. C.; and Rapp, U. R.; 1986; “The complete coding sequence of the human raf oncogene and the corresponding structure of the c-raf-1 gen.”; Nucleic Acids Res.; 14, 109) was cloned in the EcoRI interface of the plasmid SPC3.7 / 5V40 including the 3.7 kb promoter region of the human surfactant-associated protein C (SPC) (Korflhagen, T. R.; Glasser, S. W., Wert, S. E. Bruno, M. D.; Daugherty, C. C.; McNeish, J. D.; Stock, J. L.; Potter, S. S.; Whitsett, J. A.; 1990; “Cis-acting sequences from a human surfactant protein gene confer pulmonary-specific gene expression in transgenic mice.”; Proc. Natl. Acad. Sci.; 87, 6122). In analogous manne...

example 2

[0030] The linearization and the pronucleus injection were performed as follows. The transgenic vectors were cut with the restriction endonucleases NotI and NdeI, cleaned with a preparative agarose gel (Sambrooks et al., 1989, see below), and diluted to a concentration of 1 ng / ml. 200 ng of the linearized DNA fragments were injected into the male pronuclei of fertilized oocytes. Transgenic founder mice were identified by analysis of the genomic DNA isolated from tail ends by Southern Blot (see also example 3). The founder mice were crossed with non-transgenic B6D2 mice, in order to establish stable lines.

[0031] The used mice were C57BL / 6xDBA F2 mice (B6D2 mice), and were obtained from Harlan Winkelmann (Borchen) and from Charles River (Sulzfeld), and were held and bred on in the stable of the MSZ (Institut für Medizinische Strahlenkunde and Zellforschung, Worzburg University, 097078 würzburg) under pathogen-free conditions.

example 3

[0032] PCR and Southern Blot for genotypization were performed by the following operational instructions. For the detection of the transgenic integration, 10 μg genomic DNA from tail ends were cut over night with 40 units BamHI, separated on a 0.7 % agarose gel by means of electrophoresis, and transferred by means of a capillary blot to nitrocellulose (Sambrook, J.; Fritsch, E. F.; Maniatis, T.; “Molecular Cloning: a laboratory manual)”; Cold Spring Harbor Laboratory Press). After fixation of the DNA by u.v. light followed the detection of the transgene by hybridization of the membrane (Church, G. M. and Gilbert, W.; “Genomic sequencing”; Proc. Natl. Acad. Sci.; 81, 1991) with a Raf-1 probe (containing the Raf-1 / SV40 sequence) that was obtained by digestion of the transgenic vector SPC-Raf-1 with BamHI. The bound probe cross-hybridizing also with the mouse-Raf locus, was detected by exposure of the membrane on film material, and marked a 3.4 kb Raf 1 / SV40 fragment or a 1.8 kb ΔRaf (...

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Abstract

The invention relates to a transgenic non-human mammal whose cells express a constitutively active oncogenic mutant of the kinase-domain of the Raf-1 gene or a protein coded by a corresponding normal allele or derivative of the A, B, or c-Raf-1 gene.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. application Ser. No. 10 / 371,138 that claims the benefit of priority under 35 U.S.C. § 119 of 197 54 774.5 filed Nov. 28, 1997, the entire contents of each is incorporated herein by reference.FIELD OF THE INVENTION [0002] The invention relates to a transgenic non-human mammal, to a method for producing the latter, to the utilization thereof, to a cell tissue therefrom, to a method for producing such cell tissue, to the utilization thereof, to a recombinant DNA expression vector and to the utilization of such vector.—The term non-human mammal refers to taxonomically higher units than animal species. Transgenic animals are organisms carrying an additional gene not originating from their species, that is a foreign gene in their genome. For the purpose of the invention in particular, such transgenic animals are meant that have the foreign gene also in the germ cells, that is which hand on the foreig...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01K67/027C12N5/06C07K14/82C12N5/10C12N9/12C12N15/09C12N15/12C12N15/85G01N33/50
CPCA01K67/0275A01K67/0278A01K2217/05A01K2217/072A01K2227/105A01K2267/0331C07K14/82C12N9/1205C12N15/8509C12N2830/008
Inventor RAPP, ULF
Owner RAPP ULF
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