Artificial Chromosome Vector for Stable Multi-Gene Maintenance
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Solution Overview
Problem
Existing gene insertion systems, such as those using plasmids, face limitations in the number of genes that can be inserted and stability issues due to the size and properties of the inserted genes, leading to challenges in maintaining multiple gene copies and controlling gene expression.
Innovation Solution
An artificial chromosome vector derived from eukaryotic chromosomes, comprising repetitive units of ribosomal RNA genes and intergenic regions with barcode sequences for target gene insertion, autonomously replicating and incorporating multiple genes, and utilizing natural amplification mechanisms for stable maintenance and controlled expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If a target gene is inserted into the IGS2 region of rDNA, then multiple genes can be incorporated, but the cohesin binding sequence may be damaged reducing rDNA stability
Solution Approach 1:
The patent creates multiple copies of the target gene by inserting them into IGS1 regions across multiple rDNA repeats. Instead of inserting a single gene into IGS2, the system uses the natural repetition of rDNA units (each containing IGS1) to generate multiple gene copies, thereby achieving gene amplification without compromising the integrity of cohesin binding sites in IGS2.
Solution Approach 2:
The patent divides the gene incorporation strategy into separate functional regions: IGS1 is designated for target gene insertion while IGS2 is preserved for cohesin binding and stability functions. This spatial segmentation allows simultaneous achievement of gene incorporation and maintenance of rDNA stability by assigning different roles to different intergenic regions.
2Adaptability or versatility
If multiple genes are inserted using existing plasmid systems, then gene diversity increases, but stability and maintenance become problematic
Solution Approach 1:
The patent exploits the cell's own rDNA amplification machinery to maintain and replicate the inserted genes. The rDNA repeat units, which naturally undergo replication and amplification in the cell, now carry the target genes. This self-service approach allows the cellular machinery to automatically maintain multiple gene copies without requiring external plasmid maintenance systems, thereby improving stability.
Solution Approach 2:
The patent merges the target genes with the rDNA repeat structure, creating a hybrid system where exogenous genes are integrated into the endogenous rDNA amplification framework. By combining the target genes with the naturally replicating and amplifying rDNA units, the system achieves stable maintenance of multiple genes through the host's own replication mechanisms.
3Manufacturing precision
If rDNA copy number is reduced artificially, then control over gene dosage is improved, but the natural amplification mechanism restores copies reducing control
Solution Approach 1:
The patent performs preliminary action by inserting target genes into the rDNA structure before the cell's natural amplification mechanism acts. The genes are pre-positioned in the IGS1 regions of rDNA repeat units, and then the cell's amplification machinery is allowed to operate. This preliminary placement ensures that when amplification occurs, the target genes are automatically replicated along with the rDNA, achieving controlled gene dosage through the natural process.
Data Source
AI summary
Provided is an artificial chromosome vector derived from a chromosome of a eukaryote, the vector comprising repetitive units of multiple ribosomal RNA genes (rDNA) and intergenic regions between the repetitive units, wherein an intergenic region 1 (IGS1) of the intergenic regions comprises a barcode sequence for incorporating and distinguishing a target gene.