System and method for identifying erythropoietin-responsive genes

Abstract

The present invention relates to the generation of a population of Epo-responsive marrow derived cells that express Epo-responsive genes and gene products. The present invention also relates to the detection of Epo-responsive genes and gene products as well as to the detection of the administration of Epo, Epo-derivatives and Epo-mimetics in subjects.

1 . A substantially homogeneous primary erythroblast population from bone marrow which, following Epo-deprivation culture for a period of time sufficient to substantially decrease Epo receptor signaling, responds to culture in the presence of Epo by up-regulating the transcription of the Epo-responsive gene podocalyxin by more than three-fold. 
     
     
         2 . The substantially homogeneous primary erythroblast population of  claim 1 , wherein a second Epo-responsive gene is selected from one or more of oncostatin-M and growth differentiation factor-3 and wherein said oncostatin-M is upregulated by more than three-fold and said growth differentiation factor-3 is upregulated by more than 50%. 
     
     
         3 . The substantially homogeneous primary erythroblast population of  claim 1 , wherein said population expresses Kit pos /CD71 high  on the surface of the cells. 
     
     
         4 . The substantially homogeneous primary erythroblast population of  claim 1 , wherein said population is TER119 neg . 
     
     
         5 . The substantially homologous primary erythroblast population of  claim 2 , wherein said up-regulation of podocalyxin, ocostatin or growth differentiation factor-3 is detected by an immunological detection assay. 
     
     
         6 . The immunological detection assay of  claim 5 , wherein said assay is selected from a group consisting of fluorescent labeling, flow cytometry, Western blot, immuno-sandwich assay and immunodiffusion assay. 
     
     
         7 . The substantially homologous primary erythroblast population of  claim 2 , wherein said up-regulation of podocalyxin, ocostatin or growth differentiation factor-3 is detected by PCR, Northern blot or Southern blot. 
     
     
         8 . The substantially homogenous primary erythroblast population of  claim 1  wherein the conditions for culture in the presence of Epo comprise about 5 U/ml Epo for about 90 minutes. 
     
     
         9 . A method for identifying an Epo-responsive gene, the method comprising:
 a) providing a substantially homogeneous primary erythroblast population from bone marrow which, following Epo-deprivation culture for a period of time sufficient to substantially decrease Epo receptor signaling, responds to culture in the presence of Epo by up-regulating podocalyxin transcription by more than 3-fold;   b) culturing the cells of step a) in media which is substantially free of Epo for a period of time sufficient to substantially decrease Epo receptor signaling, the media comprising insulin in a range from 10 ng/ml up to 50 ng/ml;   c) culturing the cells of step b) in the presence of Epo at levels sufficient to modulate transcription of Epo-responsive genes; and   d) identifying genes which respond to the presence of Epo by increasing or decreasing transcription levels.   
     
     
         10 . The method of  claim 9 , wherein said incubation mixture further comprises transferrin. 
     
     
         11 . The method of  claim 10 , wherein said transferrin is provided at a level of between about 5 Hg/ml and 500 μg/ml. 
     
     
         12 . The method of  claim 9 , wherein said incubation mixture further comprises bovine serum albumin (BSA). 
     
     
         13 . The method of  claim 12 , wherein said BSA is provided at a level of between 0.05% and 5.0% w/v. 
     
     
         14 . A method for detecting, in vivo, the effects of exogenous Epo or derivatives or mimetics thereof, the method comprising:
 a) providing a blood sample from an individual;   b) determining the level of expression of an Epo responsive gene product on the surface of circulating erythroid cells in the blood sample of the individual;   c) comparing the level of expression determined in step b) with a standard level of expression accepted as representative of the in vivo expression level on the surface of red blood cells in the individual in the absence of exogenous Epo or derivatives or mimetics thereof, a substantial increase in the level determined in step b), as compared with the standard level of expression accepted as representative of the in vivo expression level on the surface of red blood cells in the individual in the absence of exogenous Epo or derivatives or mimetics thereof, being indicative of the effects of exogenous Epo or derivatives or mimetics thereof.   
     
     
         15 . The method of  claim 14 , wherein said Epo-responsive gene is selected from a group consisting of podocalyxin, oscosatin and growth differentiation factor-3. 
     
     
         16 . The method of  claim 14  wherein the Epo-responsive gene is podocalyxin. 
     
     
         17 . The method of  claim 14 , wherein said Epo-responsive gene is detected by an immunological technique. 
     
     
         18 . The immunological technique of  claim 17 , wherein said assay is selected from a group consisting of fluorescent labeling, flow cytometry, Western blot, immuno-sandwich assay and immunodiffusion assay. 
     
     
         19 . The method of  claim 14 , wherein said Epo-responsive gene is detected by PCR, Northern blot or Southern blot or a combination thereof. 
     
     
         20 . The method of  claim 14 , wherein said circulating erythroid cells are selected from one or more of reticulocytes, erythroblasts and red blood cells. 
     
     
         21 . The method of  claim 14 , wherein said exogenous Epo, Epo-derivatives and mimetics are selected from a group consisting of Aransep, Epo-PEG conjugates, recombinant Epo and Epo mimetics.