Immortalization of cells including neuronal cells

Abstract

The instant invention provides methods for immortalizing cells. The invention further provides immortalized cell lines, e.g., neuronal cell lines, and methods of using these cell lines in screening assays.

1 . A method for generating an immortalized human cell comprising:
 introducing into a cell a DNA segment encoding an oncogene;   selecting for a cell containing the DNA segment; and   introducing a hTERT into the selected cell;   thereby generating an immortalized cell.   
     
     
         2 . The method of  claim 1 , wherein the DNA segment is contained in a plasmid. 
     
     
         3 . The method of  claim 1 , wherein hTERT is introduced by a retrovirus. 
     
     
         4 . The method of  claim 1 , wherein the cell is a neuronal cell. 
     
     
         5 . The method of  claim 4 , wherein the neuronal cell is selected from neuronal cells from the brain, spinal cord, and dorsal root sensory ganglia. 
     
     
         6 . The method of  claim 1 , wherein the human cell is a dorsal root ganglia neuron. 
     
     
         7 . The method of  claim 6 , wherein the dorsal root ganglion neuron is a nociceptive dorsal root ganglion neuron. 
     
     
         8 . The method of  claim 1 , wherein the cell is a glial cell. 
     
     
         9 . The method of  claim 8 , wherein the glial cell is an astrocyte, oligodendrocyte or a Schwann cell. 
     
     
         10 . The method of  claim 1 , further comprises contacting the immortalized cell with an agent that causes differentiation. 
     
     
         11 . The method of  claim 10 , wherein the agent is cyclic AMP or an analog thereof or an agent that increases intracellular cAMP levels. 
     
     
         12 . The method of  claim 11 , wherein the agent is forskolin. 
     
     
         13 . A method of producing immortalized neuronal cells comprising:
 introducing a DNA segment encoding an oncogene into a neuronal cell;   selecting for neuronal cells that contain the DNA segment;   introducing hTERT into the selected cells; and   selecting for cells that contain hTERT;   thereby producing immortalized neuronal cells.   
     
     
         14 . The method of  claim 13 , wherein the DNA segment is contained in a plasmid. 
     
     
         15 . The method of  claim 13 , wherein the neuronal cells are selected from a group consisting of neuronal cells from the brain, neuronal cells from the spinal cord, dorsal root sensory ganglia, and autonomic ganglia. 
     
     
         16 . The method of  claim 13 , wherein the oncogene is selected from the group consisting of Ras, Myc, Raf, and large T-Antigen. 
     
     
         17 - 28 . (canceled) 
     
     
         29 . A method of producing immortalized dorsal root ganglion neuronal cell line comprising:
 introducing a plasmid comprising an SV40 large T-antigen into dorsal root ganglion cell;   selecting dorsal root ganglion cells that contain the plasmid;   introducing hTERT into the selected cells; and   selecting for cells that contain hTERT;   thereby producing immortalized dorsal root ganglion neuronal line.   
     
     
         30 . The method of  claim 29 , wherein hTERT is introduced by a retrovirus. 
     
     
         31 . An immortalized nociceptive dorsal root ganglion neuron. 
     
     
         32 . The immortalized nociceptive dorsal root ganglion neuron of  claim 31 , wherein the neuron expresses markers of nociceptive dorsal root ganglion neurons. 
     
     
         33 . The method of claim  24 , wherein the nociceptive dorsal root ganglion neurons express a capsaicin receptor, TRPV1, GDNF-receptor, NGF-receptor, or a sodium channel. 
     
     
         34 . The method of claim  24 , wherein the nociceptive dorsal root ganglion neurons respond to capsaicin by elevating intracellular calcium flux or generate action potentials when polarized. 
     
     
         35 . The immortalized nociceptive dorsal root ganglion neuron of  claim 33 , wherein the capsaicin receptor is TRPV1. 
     
     
         36 . The immortalized nociceptive dorsal root ganglion neuron of  claim 35 , further comprising an oncogene and hTERT. 
     
     
         37 - 40 . (canceled) 
     
     
         41 . A method of identifying a modulator of pain, comprising:
 contacting an immortalized dorsal root ganglion neuronal cell of  claim 29 , with a candidate modulator; and   determining if the candidate modulator is binds to and/or modulates the dorsal root ganglion neuronal cell;   thereby identifying a modulator of pain.