Immortalized Neuronal Cell Lines via SV40 and hTERT
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Solution Overview
Problem
Current methods are ineffective in immortalizing neuronal cells, which are difficult to transform, limiting the development of novel therapeutics for neuropathic pain and other diseases, as existing techniques have low efficiency and do not maintain the biochemical and electrophysiological properties of primary neurons.
Innovation Solution
A method involving the introduction of a DNA segment encoding an oncogene, such as the SV40 large T-antigen, and human telomerase reverse transcriptase (hTERT) into neuronal cells, followed by selection and differentiation with agents like forskolin to generate stable immortalized nociceptive dorsal root ganglion neurons that retain key markers and functions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of stationary object
If conventional immortalization methods are used on neuronal cells, then cell lines can be generated, but the biochemical and electrophysiological properties of primary neurons are lost
Solution Approach 1:
The patent changes the molecular parameters of immortalization by using specific combinations of oncogenes (SV40 large T-antigen, hTERT, hRAS) at controlled expression levels. This allows achieving cellular immortality while preserving neuronal differentiation markers and electrophysiological properties, resolving the contradiction between cell line stability and neural property retention.
Solution Approach 2:
The patent creates a composite cellular system by combining multiple genetic elements (oncogenes, telomerase, neuronal differentiation factors) within a single cell type. This composite approach enables simultaneous achievement of immortality and functional preservation, as each component contributes specific properties that collectively maintain both stability and neural characteristics.
2Duration of action of stationary object
If neuronal cells are transformed using existing techniques, then immortalized cell lines can be obtained, but the transformation efficiency is very low
Solution Approach 1:
The patent uses viral vectors as intermediaries to deliver oncogenes and other genetic elements into neuronal cells. This intermediary approach significantly enhances transformation efficiency compared to direct transfection methods, while the controlled expression systems ensure that neuronal properties are preserved during the transformation process.
Solution Approach 2:
The patent optimizes multiple parameters including MOI (multiplicity of infection), transfection conditions, and selection pressures to maximize transformation efficiency. By carefully controlling these parameters, the method achieves high productivity in generating immortalized neuronal cell lines while maintaining their functional characteristics.
3Productivity
If oncogenes are introduced to immortalize neuronal cells, then cell division capacity increases, but cellular differentiation and maturation are inhibited
Solution Approach 1:
The patent employs inducible and regulated expression systems for oncogenes, allowing dynamic control of proliferation signals. By adjusting the expression levels of oncogenes like SV40 large T-antigen and hRAS, the system can be tuned to permit cell division while simultaneously allowing differentiation programs to proceed, thus resolving the contradiction between proliferation and differentiation.
Solution Approach 2:
The patent separates the functions of immortalization and differentiation by using distinct genetic elements and regulatory mechanisms. Oncogenes provide the immortalization function while separate differentiation factors (neurotrophic factors, retinoic acid, etc.) drive maturation programs, allowing both processes to occur independently and cooperatively without mutual inhibition.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach successfully produces immortalized neuronal cell lines that maintain the biochemical and electrophysiological properties of primary neurons, enabling effective high-throughput screening for modulators of neuropathic pain and other conditions.
Implementation Method 1
The DNA segment encodes an oncogene, such as the SV40 large T-antigen
Implementation Method 2
human telomerase reverse transcriptase (hTERT) into neuronal cells
Implementation Method 3
differentiation with agents like forskolin
Data Source
AI summary
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.