Transposon Integration With Episomal Selection for Cell Proliferation
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Solution Overview
Problem
Mass production of cells for cell-based meat products is limited by factors such as senescence before reaching required density, leading to high costs and resource consumption.
Innovation Solution
A transposon system where a selection marker is located outside the 5′ and 3′ ITRs, allowing transient expression and integration of a gene of interest into the cell genome without integrating the selection marker, thereby increasing cell proliferation capacity and reducing the need for extensive passaging.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If selection markers are integrated into the cell genome for stable selection, then selection efficiency is improved, but genetic modification complexity increases and safety concerns arise
Solution Approach 1:
The selection marker is extracted from the transposon structure and placed outside the ITRs, allowing it to remain episomal while the transposon integrates. This separation enables selection functionality without permanent genomic integration of the marker, reducing genetic modification complexity and safety concerns.
Solution Approach 2:
The transposon acts as an intermediary vehicle that delivers the selection marker to the cell without requiring its integration. The transposon integrates and provides transient expression, mediating the selection process while the marker itself remains extrachromosomal and is eventually lost.
2Quantity of substance
If cells are passaged extensively to reach required density, then sufficient biomass is produced, but senescence occurs and costs increase
Solution Approach 1:
The transposon system performs preliminary integration and selection marker delivery before extensive passaging is required. This enables early selection of successfully transduced cells, allowing optimization of culture conditions and passage strategies to maximize proliferative capacity and reach required densities before senescence.
3Reliability
If selection markers remain in the final product, then selection function is maintained, but safety for consumption is compromised
Solution Approach 1:
The selection marker is extracted from the integrating transposon structure and positioned outside the ITRs, ensuring it remains episomal and is naturally lost during cell culture expansion. This eliminates the marker from the final product while maintaining its selection function during the production process.
Solution Approach 2:
The system is designed to discard the selection marker after it has served its purpose. The marker is temporarily present during selection but is naturally eliminated during subsequent passaging, allowing recovery of cells that have the desired transgene integration without the marker.
Data Source
AI summary
Provided herein are transposon/transposase systems for transposing and selecting of transposed cells where the system includes a transposase or polynucleotide encoding a transposase; and an exogenous nucleic acid sequence comprising: a transposon comprising: a 5′ inverted terminal repeat (ITR), a first polynucleotide encoding a gene of interest; and a 3′ inverted terminal repeat (ITR); and (ii) a polynucleotide encoding an at least first selection marker located outside of the 5′ ITR and 3′ ITR, where upon introducing (a) and (b) into a cell the transposon is transposed into the cell's genome and the polynucleotide encoding the selection marker is episomal.