Osteopontin knock-out mouse and methods of use thereof

Abstract

A transgenic non-human animal with alterations in the osteopontin gene is prepared by introduction of a gene encoding an altered osteopontin protein into a host non-human animal. Methods for using transgenic mice so generated to screen for agents that effect osteopontin's cellular modulating activity are also provided.

What is claimed is:  
     
         1 . A transgenic mouse harboring a homozygous null mutation in its endogenous osteopontin gene wherein said mutation has been introduced into said mouse via homologous recombination in embryonic stem cells, and further wherein said mouse does not express a functional mouse osteopontin protein.  
     
     
         2 . The transgenic mouse of  claim 1 , wherein said mouse is fertile and transmits said null mutation to its offspring.  
     
     
         3 . The transgenic mouse of  claim 1 , wherein said null mutation has been introduced into an ancestor of said mouse at an embryonic stage following microinjection of embryonic stem cells into a mouse blastocyt.  
     
     
         4 . The transgenic mouse of  claim 1 , wherein said null mutation has been introduced into an ancestor of said mouse at an embryonic stage following co-incubation of embryonic stem cells with a fertilized egg or morula.  
     
     
         5 . The transgenic mouse of  claim 1 , wherein said mutation is introduced via an insertion of a targeting DNA construct containing a neomycin cassette, into the EagI site of exon  6  of the endogenous osteopontin gene.  
     
     
         6 . A method for screening for therapeutic agents which affect osteopontin activity, comprising: 
 a) administering a test compound to the transgenic mouse of  claim 1;     b) assessing said mouse for an alteration in an osteopontin-related physiological process, said process being selected from the group consisting of bone remodeling, angiogenesis, inhibition of nitric oxide production, renal ischemic damage, atherosclerosis, monocyte differentiation, tumorigenesis, osteoporosis and osteoclastogeneis.    
     
     
         7 . A method for assessing the activity of therapeutic agents useful for the treatment and prevention of osteoporosis, comprising: 
 a) providing a pair of osteopontin knock-out mice;    b) ovariectomizing one of said pair of osteopontin knock out mice;    c) administering a therapeutic agent to said animals; and    d) assessing both animals for promotion of ovariectomized-induced osteoporosis.    
     
     
         8 . A method for screening for therapeutic agents useful for the treatment or prevention of osteoporosis, comprising: 
 a) providing a pair of wild-type mice;    b) ovariectomizing one of said pair of wild-type mice;    c) administering a therapeutic agent to said animals; and    d) assessing both animals for inhibition of ovariectomized-induced osteoporosis.    
     
     
         9 . A method for screening for therapeutic agents useful for the treatment of renal ischemia, comprising: 
 a) providing a pair of osteopontin knockout mice;    b) clamping renal arteries of said mice to induce renal ischemia in the presence of a test compound suspected of preventing renal damage; and    c) assessing said animals prevention of said renal damage mediated by said test compound.    
     
     
         10 . A method as claimed in  claim 9  wherein renal damage is assessed by measuring levels of blood urea nitrogen following reperfusion of said ischemic kidneys.  
     
     
         11 . A method as claimed in  claim 9  wherein renal damage is assessed by measuring levels of creatinine following reperfusion of said ischemic kidneys.  
     
     
         12 . A method for producing plasma protein specific antibodies, comprising: 
 a) generating a knock out mouse wherein the gene encoding the plasma protein of interest has been deleted;    b) administering an immunogenic amount of said plasma protein of interest or fragments thereof;    c) harvesting serum from said mouse; and    d) screening said serum for antibodies immunoreactive to said plasma protein.    
     
     
         13 . An antibody produced by the method of  claim 12 .  
     
     
         14 . A method for producing osteopontin-specific antibodies, comprising: 
 a) immunizing an osteopontin knock out mouse with an immunogenic amount of osteopontin or fragments thereof;    b) harvesting serum from said mouse; and    c) screening said serum for antibodies immunoreactive to osteopontin.    
     
     
         15 . An antibody preparation produced by the method of  claim 14 .  
     
     
         16 . A method for producing osteopontin-specific antibodies, comprising: 
 a) immunizing an osteopontin knock out mouse with an immunogenic amount of osteopontin or fragments thereof;    b) harvesting the spleen of said mouse and fusing said spleen cells with a myeloma cell line containing a mutation to facilitate isolation of fused spleen/myeloma cells;    c) culturing said fused cells in media containing a selection agent; and    d) screening said media for the presence of antibodies immunoreactive to osteopontin.    
     
     
         17 . A monoclonal antibody produced by the method of  claim 14 .  
     
     
         18 . An osteopontin specific monoclonal antibody selected from the group consisting of AKMZA1, AKM4AG9, AKM2C5, AKM1G4, AKM8B3, and AKM10F6.