Mouse Artificial Chromosome Vector for Stable Transgene Retention

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Solution Overview

Problem

Conventional transgenic mice have issues with random transgene insertion sites causing reduced expression, inability to control transgene copy number, and limitations in cloning large genes or gene clusters, leading to unstable retention and transmission of human artificial chromosomes in mouse cells, making detailed gene function analysis challenging.

Innovation Solution

Development of a mouse artificial chromosome vector using a naturally occurring mouse centromere, a mouse-chromosome-derived long-arm fragment with deleted endogenous genes, and a telomere sequence, which is stably retained in mammalian cells and tissues, allowing for precise and reproducible gene expression analysis.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If conventional transgene introduction methods are used, then transgenes can be introduced into mouse cells, but the insertion sites are random causing reduced expression and inability to control copy number

Engineering Contradiction:

Improvetransgene insertion precisionVSAvoidcontrol over transgene copy number

Core Design Contradiction:

Manufacturing precisionVSEase of operation

Solution Approach 1:

The invention segments the mouse chromosome into specific regions (centromere, long-arm fragment, short-arm fragment) to create an artificial chromosome vector. This segmentation allows precise control over which chromosomal regions are retained and which are deleted, enabling controlled transgene insertion without random integration. The segmented structure provides defined boundaries for gene expression control.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The mouse artificial chromosome vector acts as an intermediary carrier between the transgene and the host cell genome. Instead of direct random integration, the vector serves as a controlled intermediate that carries the transgene to specific locations, enabling precise insertion site control and manageable copy number through the vector's structured chromosomal design.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Quantity of substance

If human artificial chromosome vectors are used in mouse cells, then large genes or gene clusters can be cloned, but the vectors are not stably retained and show variable retention rates

Engineering Contradiction:

Improvesize of cloned gene or gene clusterVSAvoidstability of vector retention

Core Design Contradiction:

Quantity of substanceVSReliability

Solution Approach 1:

The invention changes the critical parameter of centromere origin from human to mouse. By using a mouse centromere instead of a human centromere in the artificial chromosome vector, the vector achieves species-matched stability in mouse cells. This parameter change (centromere origin) fundamentally improves retention stability while maintaining the capability to carry large genes and gene clusters.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention applies local quality by selectively retaining specific chromosomal regions (centromere, long-arm fragment, short-arm fragment) while deleting others. This localized retention strategy ensures stable vector behavior in mouse cells by keeping only the essential structural elements needed for stability, while the ability to insert large genes is maintained through the engineered chromosome structure.

Inventive Principle:

Principle #3Local quality

3Adaptability or versatility

If conventional gene transfer methods are used, then transgenes can be introduced, but the positional effect of insertion sites causes reduced expression and limits cloning size to about 200 kb

Engineering Contradiction:

Improvecloning capacityVSAvoidtransgene expression level

Core Design Contradiction:

Adaptability or versatilityVSManufacturing precision

Solution Approach 1:

The invention transitions from two-dimensional plasmid-based gene transfer to three-dimensional chromosomal integration. By utilizing the three-dimensional structure of chromosomes and integrating transgenes into specific chromosomal locations rather than relying on plasmid circular DNA, the system achieves both large cloning capacity (beyond 200 kb) and precise expression control through the spatial organization of chromosomal regions.

Inventive Principle:

Principle #17Another dimension (Dimensionality change)

Data Source

PatentUS9775331B2Rodent comprising mouse artificial chromosome vector

Publication Date: 2017.10.03 TRANS CHROMOSOMICS INC

AI summary

Disclosed is a mouse artificial chromosome vector, comprising: a natural centromere derived from a mouse chromosome; a mouse-chromosome-derived long-arm fragment formed by deleting a long-arm distal region at a mouse chromosome long-arm site proximal to the centromere; and a telomere sequence, wherein the vector is stably retained in a cell and/or tissue of a mammal. In addition, disclosed are cells or non-human animals comprising the vector, and use of the cells or non-human animals.