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

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...

AI Technical Summary

Benefits of technology

[0012] Advantageously, the foreign DNA in addition contains a promoter for the surfactant protein C, preferably for the human surfactant protein C, and this promoter is arranged in the foreign DNA with the proviso that by the promoter the transcription of the mutant of the kinase-domain of the c-Raf-1 gene or of a corresponding normal allele or a derivative of the A, B or c-Raf-1 gene is controlled in other words, the promoter being in a natural manner arranged ahead of the gene coding the surfactant protein C, is arranged instead, according to the invention, at a suitable position of the mutant or of the gene. The precise arrangement of promoter and mutant or gene with regard to each other is usual knowledge of the man skilled in the art. If the exact positioning of the special promoter ahead of the gene utilized according to the invention cannot be derived from basic considerations, simple tests with different variants of positioning can however be performed, in order to determine a suitable position: The number of variants in question, under consideration of the general technical knowledge, is however very limited. The surfactant protein C plays a role for the surfactant factor reducing the alveolar surface tension between the lung epithelium and air and thus preventing that the alveoli will collapse during breathing-out and that the epithelia will stick together. By application of the promoter for the surfactant protein C it is achieved that only the transcription factors specifically or with increased frequency occurring in the lung and inter-reacting with this promoter can so to speak switch on the mutant, with the result that lung tumors will be formed at high selectivity and reproducibility.

[0014] A transgenic non-human mammal as described above is obtainable by the following steps: a) integration of the cDNA sequence of a constitutively active oncogenic mutant of the kinase-domain of the c-Raf-1 gene or of a corresponding normal allele or a derivative of the A, B or cRaf-1 gene in an expression vector, b) insertion of the transgenic vector obtained in step a), preferably after linearization, in pronuclei of fertilized oocytes from a non-human mammal, c) implantation of the oocytes obtained in step b) in brood animals of the same species as the donor species of the oocytes and delivery of descendant animals from the oocytes, d) genotypization and selection of the descendant animals obtained in step c) with the proviso that cells of the selected mammals express a constitutively active oncogenic mutant of the kinase-domain of the c-Raf-1 gene or a protein coded by a corresponding normal allele or a derivative of the A, B or c-Raf-1 gene. Genotypization can be obtained by means of methods well known to the man skilled in the art, for instance by tail biopsy by means of PCR (polymerase chain reaction, a method for the in-vitro amplification of a defined DNA fragment) and Southern Blot (a method for the analysis of DNA fragments in DNA preparations), with those mammals being selected whose cells can be proven by the examinations of the genotypization to contain the foreign DNA according to the invention. Advantageously, the expression vector used is step a) contains a promoter for the surfactant protein C, preferably a promoter for the human surfactant protein C. This promoter is arranged in the foreign DNA with the proviso that by the promoter the transcription of the mutant of the kinase-domain of the Raf-1 gene or of a corresponding normal allele or derivative of the A, R or c-Raf-1 gene is controlled. In detail, the foreign DNA may comprise for instance either the healthy (FIG. 1) or a constitutively active oncogenic mutant of the Raf-1 gene with a sequence according to one of FIG. 1, however with deletion as ΔRaf (26-302). In place of a deletion, point mutations of the sequence shown in FIG. 1 can also be used. Such point mutations can for instance specifically be caused by administration of 1-ethyl-1-nitrosourea (ENU) to animals having the healthy sequence, thereby such mutants being accessible in a simple manner

[0015] The invention also relates to a method for producing a transgenic non-human mammal as described above according to patent claims 10 and 11. Non-human mammals according to the invention are used for pre-clinic examinations of the effectiveness of substances directed against lung carcinomas and / or therapeutical methods directed against lung carcinomas, in particular for the pre-clinic examination of the effectiveness of substances inhibiting Raf-kinase. Such substances completely inhibit or reduce the activity of Raf-kinases, thereby possibly a means for the deactivation particularly of Raf-oncoproteins and therefore for the proliferation inhibition of tumor cells being found. Another advantageous utilization of a non-human, mammal according to the invention is the investigation of the pathogenesis of lung tumors, thereby a better understanding of the disease per se being possible.

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.

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