Cell Immortalization via hTert hRb hE2F1 Gene Combinations
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Solution Overview
Problem
Current methods for immortalizing cells are limited by their inability to produce biologically relevant cell lines that reflect in vivo properties, as they often result in drastically altered or mutated cell lines, and lack universality across different cell types and species, with existing immortalization techniques inducing genomic instability and requiring specific genes for specific cell types.
Innovation Solution
A method involving the use of vectors that introduce genes such as Id2, Nanog, and Fos or c-Myc to activate BMP signaling, maintain pluripotency, and facilitate cell cycle progression, allowing for the immortalization of cells while retaining their differentiation-specific properties, and optionally including genes for cell selection, with the ability to regulate gene expression using systems like cre recombinase or tetracycline-regulated promoters.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional immortalisation regimens using known immortalising genes like SV40 large T antigen or viral oncogenes are used, then cell proliferation capacity is improved, but biological relevance is lost due to drastic alteration of cell physiology
Solution Approach 1:
The patent changes the parameters by using a specific combination of immortalising genes (hTert, hRb, hE2F1) at controlled expression levels that enable unlimited proliferation while maintaining physiological relevance. The expression levels are carefully regulated to avoid the drastic alterations caused by conventional high-level expression of single oncogenes.
Solution Approach 2:
The patent employs a composite approach by combining multiple immortalising genes (hTert, hRb, hE2F1) working together in a coordinated manner. This composite gene system achieves immortalisation more gently than single genes, preserving cellular characteristics and biological relevance while enabling unlimited proliferation.
2Ease of operation
If spontaneous immortalisation of primary cell material is used, then cell lines are easily maintained and expanded, but the success rate is low and huge amounts of primary cell material are required
Solution Approach 1:
The patent applies preliminary action by pre-selecting and transducing primary cells with the immortalising gene combination before they undergo spontaneous immortalisation. This preliminary genetic modification increases the success rate significantly, allowing immortalisation to occur at lower passage numbers and with fewer primary cells required.
Solution Approach 2:
The patent incorporates feedback mechanisms through selectable markers (e.g., GFP, antibiotic resistance genes) that provide real-time information about successful transduction and immortalisation. This feedback allows for efficient selection and expansion of immortalised cells, improving both success rate and ease of operation.
3Adaptability or versatility
If hTert is used for immortalisation, then expansion of a wide variety of cell types is achieved, but cell type-specific genes are required for efficient immortalisation of certain cell types
Solution Approach 1:
The patent achieves universality by creating a core immortalising gene system (hTert, hRb, hE2F1) that functions across diverse cell types. While cell type-specific genes may still be needed for optimal efficiency, this universal core system dramatically broadens the range of immortalisable cell types compared to using hTert alone or cell type-specific approaches.
Solution Approach 2:
The patent segments the immortalisation system into a universal core component (hTert, hRb, hE2F1 combination that works across cell types) and cell type-specific optional components. This segmentation allows the universal system to provide broad adaptability while minimizing the total number of genes required by only adding cell type-specific genes when necessary for efficiency.
4Reliability
If primary cells are used, then high biological relevance is achieved, but proliferation capacity is limited and expansion is restricted
Solution Approach 1:
The patent changes the proliferation parameters of primary cells by introducing controlled expression of immortalising genes (hTert, hRb, hE2F1). This parameter change enables unlimited proliferation while maintaining the biological relevance of primary cells, as the genes are expressed at physiological levels that preserve cellular characteristics.
Solution Approach 2:
The patent uses the immortalising gene combination as an intermediary that bridges the gap between primary cells (high biological relevance, limited proliferation) and conventional cell lines (unlimited proliferation, low biological relevance). The intermediaries (hTert, hRb, hE2F1) enable primary cells to achieve unlimited proliferation while retaining their physiological properties.
Data Source
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AI summary
The present invention relates to a method and to vectors for the immortalisation of cells independent of their type. It further relates to a cell or a cell line produced with the method or the vectors of the invention. The invention also relates to the use of this cell or cell line in in vitro applications and in the treatment of disease.