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Immortalization of cells including neuronal cells

a neuronal cell and immortalization technology, applied in the field of immortalization of cells including neuronal cells, can solve the problems of unsatisfactory treatment of neuropathic pain, limited clinical application prospects, and limited clinical application prospects, and achieve the effect of increasing intracellular camp levels

Inactive Publication Date: 2009-12-03
THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent is about methods for making immortalized cells, specifically neurons, from difficult to immortalize cells such as brain and spinal cord neurons. The methods involve introducing a DNA segment encoding an oncogene and a plasmid containing hTERT into the cells, and selecting cells that contain the DNA and hTERT. The immortalized cells can also be contacted with an agent that causes differentiation, such as cyclic AMP or forskolin. The immortalized cells maintain the biochemical and electrophysiological properties of primary neurons and can express a capsaicin receptor, TRPV1, GDNF-receptor, NGF-receptor, or a sodium channel. The invention also provides immortalized nociceptive dorsal root ganglion neurons that maintain the biochemical and electrophysiological properties of primary neurons and express a capsaicin receptor, TRPV1.

Problems solved by technology

Although the American Pain Society estimates that nearly 50 million Americans are totally or partially disabled by pain, there are currently very few effective, well-tolerated treatments available (Wetzel et al.
Indeed, existing therapeutics cause a range of undesirable side effects primarily due to the difficulty in developing small-molecule drugs capable of specifically targeting the receptor / channel of choice.
However, treatment of neuropathic pain is often unsatisfactory; persistent neuropathic pain affects quality of life and lead to significant morbidity.
To date, attempts to immortalize neuronal cell lines have achieved little success.
Also, a temperature sensitive mutant T antigen has been used to immortalize neuronal populations, but the efficiency of this technique has been very low (Eves (1994) Brain Res 656:396-404).

Method used

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  • Immortalization of cells including neuronal cells
  • Immortalization of cells including neuronal cells
  • Immortalization of cells including neuronal cells

Examples

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example 1

Generation and Characterization of DRG Neuronal Cell Line

[0067]Materials and Methods

[0068]Unless noted otherwise all reagents and materials were purchased from Invitrogen (Carlsbad, Calif.). Animal studies were conducted according the protocols approved by the institutional Animal Care and Use Committee.

[0069]Construction of SV40 Large T-Antigen and hTERT Expression Vectors

[0070]For cloning of the SV40 large T-antigen, plasmid construct pZipSV776-1 was used as the template for PCR amplification of the gene fragment coding for SV40 large T-antigen. PCR reaction was primed by oligonucleotides 5′-CACCGCTTTGCAAAGATGGATAAAG (sense) and 5′-AATTGCATTCATTTTATGTTTCA (anti-sense). Amplification was performed using the Expend High Fidelity PCR System (Roche, Indianapolis, Ind.). The PCR product was cloned into the pENTR / D-TOPO vector by directional TA-cloning. After confirmation of the sequence, the target SV40 large T-antigen gene was transferred into pLenti6 / V5-Dest vector using Gateway tech...

example 2

Generation and Characterization of Human Fetal Astrocytes and Schwann Cells

[0098]Human astrocytes or Schwann cells were prepared from aborted fetal tissues using standard cell culture techniques. Cells were cultured in Neurobasal media (Neurobasal MEM plus 10% FBS, 0.5 uM glutamin, 2% glucose, 1×B27 supplement) for 3-7 days in an incubator with 5% CO2 at 37° C. Cells were detached with a cell scraper and washed 2 times in Opti-MEM (Invitrogen, CA) and 0.8-1.0×106 cells were suspended in 100 ul Opti-MEM and dissociated with pipetting using a 200 μl tip.

[0099]Five μg pLenti6 / V5-DEST plasmid carrying hTERT gene (pLenti6 / hTERT) in 1.7 μl TE (pH8.0) and 15 μg pLenti6 / V5-DEST plasmid carrying SV40 large T antigen gene (pLenti6 / SV40) in 5 μl TE (pH8.0) were mixed with cells, transferred into a 0.2 cm gene-pulser cuvette (Bio-Rad, CA) and incubated for 5 minutes at room temperature. Gene Pulser Xcell Electroporation System (Bio-Rad, CA) was set at 850 μF×90V and the cells were pulsed once (...

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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.

Description

RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Application No. 60 / 671,865, filed 15 Apr. 2005, the entire contents of which are incorporated herein by reference.GOVERNMENT SUPPORT[0002]The following invention was supported at least in part by NIH Grants RO1 NS43991 and PO1 MH70056. Accordingly, the government may have certain rights in the invention.BACKGROUND OF THE INVENTION[0003]Neuropathic pain, also referred to as a chronic pain, is a complex disorder resulting from injury to the nerve, spinal cord or brain. There is evidence that nerve fibers in subjects with neuropathic pain develop abnormal excitability, particularly hyper-excitability, Zimmerman (2001) Eur J Pharmacol 429 (1-3):23-37. Although the American Pain Society estimates that nearly 50 million Americans are totally or partially disabled by pain, there are currently very few effective, well-tolerated treatments available (Wetzel et al. (1997) Ann Pharmacother 31 (9):1082-3). Indeed,...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/53C12N15/87C12N5/00
CPCC12N9/1276C12N2799/027C12N2510/04C12N5/0619G01N33/5058G01N2500/10
Inventor HOKE, AHMETCHEN, WEIRAN
Owner THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE
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