Transgenic mammals lacking expression of erythropoietin or of erythropoietin receptor, transgenic mammals expressing chimeric erythropoietin receptors, constructs for producing the transgenic mammals and uses therefor

Abstract

Transgenic nonhuman mammals, such as transgenic mice, which lack erythropoietin expression, in which the erythropoietin receptor is deleted, which carry a heterologous erythropoietin receptor (e.g., a chimeric receptor); constructs useful for producing such transgenic nonhuman mammals, embryonic stem cells containing the constructs, a method of producing the transgenic nonhuman mammals and a method of identifying erythropoietin mimics or mimetics.

We claim:  
     
         1 . A transgenic nonhuman mammal wherein expression of erythropoietin is lacking.  
     
     
         2 . The transgenic nonhuman mammal of  claim 1  which is a transgenic mouse.  
     
     
         3 . The transgenic mouse of  claim 2  which is a knockout mouse wherein the erythropoietin gene was deleted by homologous recombination between the mouse erythropoietin gene and DNA comprising the 2.5 Kb PstI-PstI fragment of the 5′ end of the mouse erythropoietin gene and the 5.0 Kb HindIII-KpnI fragment of the mouse erythropoietin gene.  
     
     
         4 . The transgenic mouse of  claim 2  wherein the erythropoietin receptor is deleted.  
     
     
         5 . The transgenic mouse of  claim 4  derived from a cross between a transgenic mouse lacking erythropoietin expression and a transgenic mouse wherein the erythropoietin receptor is deleted.  
     
     
         6 . A transgenic nonhuman mammal wherein the erythropoietin receptor is deleted.  
     
     
         7 . The transgenic mammal of  claim 6  which is a transgenic mouse.  
     
     
         8 . The transgenic mouse of  claim 7  which is a knockout mouse wherein the erythropoietin receptor was deleted by homologous recombination between the mouse erythropoietin receptor gene and DNA comprising the 4 Kb SalI-BamHI fragment derived from the 5′ end of the mouse erythropoietin receptor gene and the 2.7 Kb XbaI-EcoRI fragment derived from the 3′ end of the mouse erythropoietin receptor gene.  
     
     
         9 . The transgenic mouse of  claim 8  wherein expression of erythropoietin is lacking.  
     
     
         10 . A transgenic nonhuman mammal wherein expression of erythropoietin is lacking and the erythropoietin receptor is deleted.  
     
     
         11 . A transgenic nonhuman mammal wherein the endogenous erythropoietin receptor is replaced by a chimeric erythropoietin receptor.  
     
     
         12 . The transgenic nonhuman mammal of  claim 11  which is a transgenic mouse, wherein the chimeric erythropoietin receptor is a human/mouse erythropoietin receptor.  
     
     
         13 . The transgenic mouse of  claim 12  wherein the human/mouse erythropoietin receptor comprises an extracellular domain, a transmembrane domain and an intracellular domain, wherein the extracellular domain is the human erythropoietin receptor extracellular domain, the transmembrane domain is the mouse erythropoietin receptor transmembrane domain or the human erythropoietin receptor transmembrane domain and the intracellular domain is the mouse erythropoietin receptor intracellular domain.  
     
     
         14 . A DNA construct comprising: a) the 2.5 Kb PstI-PstI fragment of the 5′ end of the erythropoietin gene, b) DNA encoding a selectable marker and c) the 5.0 Kb HindIII-KpnI fragment of the erythropoietin gene, wherein the DNA encoding the selectable marker is positioned between the fragment of a) and the fragment of c).  
     
     
         15 . A DNA construct comprising: a) the 4 Kb SalI-BamHI fragment derived from the 5′ end of the mouse erythropoietin receptor gene, b) DNA encoding a selectable marker and c) the 2.7 Kb XbaI-EcoRI fragment derived from the 3′ end of the mouse erythropoietin receptor gene, wherein the DNA encoding the selectable marker is positioned between the fragment of a) and the fragment of c).  
     
     
         16 . The plasmid comprising pEPO-M1.  
     
     
         17 . The plasmid comprising pEPOR-M1.  
     
     
         18 . Mouse embryonic stem cells containing the DNA construct of  claim 14 .  
     
     
         19 . Mouse embryonic stem cells containing pEPO-M1.  
     
     
         20 . Mouse embryonic stem cells containing the DNA construct of  claim 15 .  
     
     
         21 . Mouse embryonic stem cells containing pEPOR-M1.  
     
     
         22 . A method of identifying an agent that is active in stimulating human EPO receptor, which comprises: 
 a) administering an agent to a transgenic mammal, wherein said mammal carries an EPO receptor with a human extracellular domain; and    b) assessing the effect of the agent on erythropoiesis,    wherein if the agent stimulates production of red blood cells, then the agent is active in stimulating human EPO receptor.    
     
     
         23 . A method of identifying an agent which is an erythropoietin mimic or mimetic, comprising the steps of: 
 a) administering an agent to a transgenic mouse, wherein the transgenic mouse lacks erythropoietin and carries a chimeric erythropoietin receptor comprising a human erythropoietin receptor extracellular domain, a mouse erythropoietin receptor transmembrane domain and a mouse erythropoietin intracellular domain; and    b) assessing the effect of the agent on erythropoiesis,    wherein if erythropoiesis is stimulated, the agent is an erythropoietin mimic or mimetic.