Sequential Nucleic Acid Loading via Recombination Marker Recycling
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Solution Overview
Problem
Current methods for loading multiple nucleic acids onto mammalian synthetic chromosomes are limited, and there is no efficient way to recycle drug resistance markers, which can lead to host immune responses and limitations in bioengineering applications.
Innovation Solution
The method involves using site-directed recombination to sequentially load multiple delivery vectors onto an autonomously replicating nucleic acid with a single selectable marker, allowing for the integration and excision of marker genes, enabling the reuse of these markers for multiple loadings without the need for drug selection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If drug resistance markers are used for selection, then selectable markers can be used for multiple loadings, but host immune responses may occur and drug selection is required
Solution Approach 1:
The patent extracts and removes the drug resistance marker genes from the final synthetic chromosome after they have served their selection purpose. This allows the markers to be recycled for multiple loading iterations without remaining in the host genome, thereby eliminating persistent immune responses while maintaining marker recyclability.
Solution Approach 2:
The patent implements dynamic control of marker gene presence through conditional recombination systems. The markers can be introduced, excised, and re-introduced in a controlled manner across multiple cycles, transforming from static permanent markers to dynamic recyclable elements that adapt to different loading stages.
2Quantity of substance
If multiple delivery vectors are loaded sequentially, then multiple nucleic acids can be delivered, but current methods are limited and complex
Solution Approach 1:
The patent creates a universal loading system where a single selectable marker can facilitate the sequential loading of multiple different delivery vectors. The same marker gene serves multiple functions across different loading iterations, simplifying the overall process compared to requiring unique markers for each nucleic acid delivery.
Solution Approach 2:
The patent establishes a continuous cycle where marker genes are excised after serving their selection function, and the same markers are reused in subsequent loading cycles. This continuous recycling eliminates the need to generate new markers for each delivery, streamlining the process for loading multiple nucleic acids.
3Productivity
If drug selection is used, then selectable markers can be recycled, but the process requires drugs and may have limitations
Solution Approach 1:
The patent replaces the chemical drug selection mechanism with a molecular biology-based recombination system. Instead of using drugs to select for marker-containing cells, the invention uses site-specific recombination enzymes to precisely control marker excision and recycling, eliminating the need for continuous drug treatment while maintaining high efficiency in marker recycling.
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 allows for efficient and unlimited recycling of marker genes, reducing host immune responses and enabling the co-delivery of multiple nucleic acids, thereby enhancing the bioengineering of synthetic chromosomes.
Implementation Method 1
integrating a first delivery vector comprising a marker gene and a drug resistant gene and a first nucleic acids of interest onto an autonomously replicating nucleic acid using a first recombination system
Implementation Method 2
excising the marker gene and the drug resistant gene from the autonomously replicating nucleic acid using a second recombination system with signal sites alpha
Data Source
AI summary
The present invention provides a novel method for targeted integration of nucleic acids onto an autonomously replicating nucleic acid using site-directed recombination that allows for sequential loading of multiple delivery vectors using a single selectable marker.