Metastasis models using green fluorescent protein (GFP) as a marker

Abstract

A method to follow the progression of metastasis of a primary tumor, which method comprises removing fresh organ tissues from a vertebrate subject which has been modified to contain tumor cells that express GFP and observing the excised tissues for the presence of fluorescence is disclosed. The fluorescence can also be monitored by observing the tissues in situ. Vertebrate subjects which contain GFP producing tumors are useful models to study the mechanism of metastasis, as well as to evaluate candidate protocols and drugs. In addition, subjects already harboring tumors can be treated so as to modify the endogenous tumors to contain GFP. This permits clinical applications. Finally, by injecting a contrast dye into a subject harboring a GFP-labeled tumor, angiogenesis in the tumor can be observed directly.

1 . A non-invasive method for detecting a transformed, eucaryotic cell in a mammalian subject, comprising: 
 administering to the subject a eucaryotic cell transformed with a heterologous gene encoding a fluorescent protein, wherein said subject comprises opaque tissue, and    measuring photon emission through opaque tissue of said subject wherein said photon emission is mediated by fluorescent protein expressed from said heterologous gene:    
     
     
         2 . The method of  claim 1 , wherein the measuring is done using a photodetector device.  
     
     
         3 . The method of  claim 1 , further comprising constructing a photon emission image from said measured photon emission.  
     
     
         4 . The method of  claim 3 , further comprising acquiring a reflected light image of the subject, and 
 superimposing said image of photon emission on said reflected light image to form a composite image.    
     
     
         5 . The method of  claim 2 , wherein said measuring is carried out with an intensified charge-coupled photodetector device.  
     
     
         6 . The method of  claim 2 , wherein said measuring is carried out with a cooled charge-coupled photodetector device.  
     
     
         7 . The method of  claim 1 , wherein said measuring is carried out using fiber optic cables.  
     
     
         8 . The method of  claim 7 , wherein said fiber optic cables terminate in a tightly-packed array.  
     
     
         9 . The method of  claim 7 , wherein said fiber optic cables detect light from a limited defined region of the subject.  
     
     
         10 . The method of  claim 1 , wherein said measuring consists of measuring photon emission from within the subject with a photodetector device located outside of the subject.  
     
     
         11 . The method of  claim 1 , wherein photons which make up said photon emission are visible light photons.  
     
     
         12 . The method of  claim 1 , wherein expression of the heterologous gene is regulated by an inducible promoter.  
     
     
         13 . The method of  claim 1 , wherein said eucaryotic cell is a tumor cell.  
     
     
         14 . The method of  claim 1 , wherein said eukaryotic cell is selected from the group consisting of primary culture cells, somatic cells, and lymphatic cells.  
     
     
         15 . The method of  claim 12 , wherein said promoter is a Tet promoter.  
     
     
         16 . The method of  claim 1 , wherein expression of said heterologous gene is mediated by a constitutively active promoter.  
     
     
         17 . The method of  claim 16 , wherein said constitutively active promoter is a CMV or SV40 promoter.  
     
     
         18 . The method of  claim 1 , wherein said fluorescent protein is selected from the group consisting of green fluorescent protein, lumazine, and yellow fluorescent protein.  
     
     
         19 . The method of  claim 1 , wherein a laser is used to excite the fluorescent protein.  
     
     
         20 . The method of  claim 1 , farther comprising: 
 repeating said measuring at selected time intervals,    wherein said repeating is effective to track localization of the eucaryotic cell in the subject over time.    
     
     
         21 . The method of  claim 1 , further comprising 
 administering a compound to said subject, and    measuring photon emission from said subject after administration of said compound.    
     
     
         22 . The method of  claim 21 , further comprising: 
 repeating at selected time intervals said measuring after administration of said compound,    wherein said repeating is effective to track an effect of said compound on a level of said eucaryotic cell in said subject over time.    
     
     
         23 . The method of  claim 10 , wherein the measuring is done using a photodetector device.  
     
     
         24 . The method of  claim 23 , wherein said measuring is carried out with an intensified charge-coupled photodetector device.  
     
     
         25 . The method of  claim 23 , wherein said measuring is carried out with a cooled charge-coupled photodetector device.  
     
     
         26 . The method of  claim 10 , wherein said measuring is carried out using fiber optic cables.  
     
     
         27 . The method of  claim 10 , wherein photons which make up said photon emission are visible light photons.  
     
     
         28 . The method of  claim 10 , further comprising constructing a photon emission image from said measured photon emission.  
     
     
         29 . The method of  claim 28 , further comprising 
 acquiring a reflected light image of the subject, and    superimposing said image of photon emission on said reflected light image to form a composite image.    
     
     
         30 . The method of  claim 10 , wherein said eucaryotic cell is a tumor cell.  
     
     
         31 . The method of  claim 10 , wherein said eukaryotic cell is selected from the group consisting of primary culture cells, somatic cells, and lymphatic cells.  
     
     
         32 . The method of  claim 10 , wherein said fluorescent protein is selected from the group consisting of green fluorescent protein, lumazine, and yellow fluorescent protein.  
     
     
         33 . The method of  claim 10 , wherein a laser is used to excite the fluorescent protein.  
     
     
         34 . The method of  claim 10 , wherein expression of said heterologous gene is regulated by an inducible promoter.  
     
     
         35 . The method of  claim 34 , wherein said promoter is a Tet promoter.  
     
     
         36 . The method of  claim 10 , wherein expression of said heterologous gene is mediated by a constitutively active promoter.  
     
     
         37 . The method of  claim 36 , wherein said constitutively active promoter is a CMV or SV40 promoter.  
     
     
         38 . The method of  claim 10 , further comprising: 
 repeating said measuring at selected time intervals,    wherein said repeating is effective to track localization of the eucaryotic cell in the subject over time.    
     
     
         39 . The method of  claim 10 , further comprising: 
 administering a compound to said subject, and    measuring photon emission from said subject after administration of said compound.    
     
     
         40 . The method of  claim 39 , further comprising: 
 repeating at selected time intervals said measuring after administration of said compound,    wherein said repeating is effective to track an effect of said compound on a level of said eucaryotic cell in said subject over time.