Transgenic non-human mammal with an oncogenic mutant of the c-Raf-1 gene

Abstract

The invention relates to a transgenic non-human mammal whose cells express a constitutively active oncogenic mutantof 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.

What is claimed is:  
     
         1 . A transgenic mouse whose cells 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 of the Raf gene or a derivative of the A Raf gene, the B Raf gene or the c-Raf-1 gene.  
     
     
         2 . The transgenic mouse according to  claim 1 , wherein the expression takes place in lung cells.  
     
     
         3 . A transgenic mouse, wherein the mouse contains a foreign DNA having a constitutively active oncogenic mutant of the kinase-domain of the c-Raf-1 gene or with a corresponding normal allele of the Raf gene or a derivative of the A Raf gene, the B Raf gene or the c-Raf-1 gene.  
     
     
         4 . The transgenic mouse according to  claim 3 , wherein the foreign DNA in addition contains a promoter for the surfactant protein C and wherein 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 of the Raf gene or a derivative of the A Raf gene, the B Raf gene or the c-Raf-1 gene is controlled.  
     
     
         5 . The transgenic mouse according to  claim 3 , wherein the foreign DNA in addition contains DNA of the SV40 virus.  
     
     
         6 . A transgenic mouse, which 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 of the Raf gene or a derivative of the A Raf gene, the B Raf gene or the c-Raf-1 gene in an expression vector to provide an expression vector carrying a foreign DNA,    b) insertion of the transgenic vector obtained in step a) in pronuclei of fertilized oocytes from a mouse,    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 mice 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 of the Raf gene or a derivative of the A Raf gene, the B Raf gene or the c-Raf-1 gene.    
     
     
         7 . The transgenic mouse according to  claim 6 , wherein the expression vector used in step a) contains a promoter for the surfactant protein C and wherein 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 of the Raf gene or a derivative of the A, B or c-Raf-1 gene is controlled.  
     
     
         8 . The transgenic mouse according to  claim 3 , wherein the constitutively active oncogenic mutant of the c-Raf-1 gene comprises a sequence according to FIG. 1 or a sequence ΔRaf(26-302) derived therefrom.  
     
     
         9 . A method for producing a transgenic mouse, including 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 of the Raf gene or a derivative of the A Raf gene, the B Raf gene or the c-Raf-1 gene in an expression vector to provide an expression vector carrying a foreign DNA,    b) insertion of the transgenic vector obtained in step a) in pronuclei of fertilized oocytes from a mouse,    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 mice 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 of the Raf gene or a derivative of the A Raf gene, the B Raf gene or the c-Raf-1 gene.    
     
     
         10 . The method according to  claim 9 , wherein the expression vector used in step a) contains a promoter for the surfactant protein C and wherein 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 of the Raf gene or a derivative of the A; B or c-Raf-1 gene is controlled.  
     
     
         11 . The utilization of a mouse according to  claim 1  for the pre-clinic examination of the effectiveness of substances intended against lung carcinomas and/or of therapeutical methods intended against lung carcinomas.  
     
     
         12 . The utilization according to  claim 11  for the preclinic examination of the effectiveness of substances inhibiting Rat-kinase,  
     
     
         13 . A lung cell tissue from a transgenic mouse according  claim 1 , which cell tissue has a higher probability of forming lung tumors.  
     
     
         14 . A method for producing cell tissue from a transgenic mouse, including 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 of the Raf gene or a derivative of the A Raf gene, the B Raf gene or the c-Raf-1 gene in an expression vector to provide an expression vector carrying a foreign DNA,    b) insertion of the transgenic vector obtained in step a) in pronuclei of fertilized oocytes from a mouse,    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 mice 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 of the Raf gene or a derivative of the A Raf gene, the B Raf gene or the c-Raf-1 gene,    e) removal of cell tissue from the mouse.    
     
     
         15 . The method according to  claim 14 , wherein the expression vector used in step a) contains a promoter for the surfactant protein C and wherein 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 of the Raf gene or a derivative of the A Raf gene, the B Raf gene or the c-Raf-1 gene is controlled.  
     
     
         16 . The utilization of a cell tissue according to  claim 13  for the pre-clinic examination of the effectiveness of substances intended against lung carcinomas and/or of therapeutical methods intended against lung carcinomas.  
     
     
         17 . The utilization according to  claim 16  for the preclinic examination of the effectiveness of substances inhibiting Raf-kinase.  
     
     
         18 . The transgenic mouse according to  claim 4 , wherein the promoter for the surfactant protein C is for the human surfactant protein C.  
     
     
         19 . The transgenic mouse according to  claim 6 , wherein insertion of the transgenic vector obtained in step occurs after linearization.  
     
     
         20 . The transgenic mouse according to  claim 6 , wherein the promoter for the surfactant protein C is for the human surfactant protein C.  
     
     
         21 . The method according to  claim 9 , wherein the promoter for the surfactant protein C is for the human surfactant protein C.  
     
     
         22 . The method according to  claim 9 , wherein the expression vector used in step a) in addition contains DNA of the SV40 virus.  
     
     
         23 . The method according to  claim 14 , further comprising cultivation of the removed cell tissue.  
     
     
         24 . The utilization of a non-human mammal according to  claim 1  for the examination of the pathogenesis of lung tumors.  
     
     
         25 . The utilization of a cell tissue according to  claim 13  for the examination of the pathogenesis of lung tumors.  
     
     
         26 . A recombinant DNA expression vector containing 
 a) the DNA 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 c-Raf-1 gene,    b) a promoter domain for the surfactant protein C, by means of which the transcription of the DNA sequence defined in a) is controllable,    c) as an option the DNA sequence of the SV 40 virus.    
     
     
         27 . A recombinant DNA expression vector according to  claim 26 , wherein the DNA sequence defined in a) is a sequence according to FIG. 1 or a sequence ΔRaf (26-302) derived therefrom, and/or wherein the promoter domain defined in b) is a promoter domain for the human surfactant protein c.  
     
     
         28 . The utilization of a recombinant DNA expression vector according to  claim 26  for producing a transgenic mammal or a cell tissue from such a mammal.