Exon-Humanized Mouse Design for Normal Tissue-Specific Expression
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Solution Overview
Problem
Existing methods for introducing human genes into mice often result in variable expression levels and tissue specificity, limiting their use as models for human disease therapies.
Innovation Solution
The development of exon-humanized mice with mouse introns and human exons, using CRISPR/Cas9 and donor vectors to replace mouse exons with human exons in target genes, ensuring normal expression levels and tissue specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If an isolated human gene is injected into mouse fertilized eggs to produce transgenic mice, then human gene expression is achieved, but the expression level and tissue specificity vary abnormally
Solution Approach 1:
The patent applies local quality by replacing only the exon regions of the mouse gene with human gene sequences while retaining the mouse introns and regulatory elements. This localized substitution ensures that the human coding sequences are expressed under the control of native mouse regulatory regions, achieving normal expression levels and tissue specificity without the variability associated with random transgenic integration
Solution Approach 2:
The patent segments the gene into exons and introns, selectively humanizing only the exon portions while maintaining mouse intronic sequences. This segmentation allows precise control over which parts of the gene are modified, enabling the human protein coding sequences to be expressed with proper regulation from the native mouse gene structure
2Reliability
If homologous recombination is used to knock-in human cDNA or minigene into mouse gene locus, then human gene expression is achieved, but the expression pattern varies from case to case
Solution Approach 1:
The patent uses local quality by targeting only the exon regions for humanization while preserving the native mouse introns and regulatory elements. This approach ensures consistent expression patterns across different experiments because the endogenous regulatory machinery remains intact, eliminating the variability associated with different minigene constructions
Solution Approach 2:
The patent creates a modified copy of the native gene where only the exon sequences are replaced with human sequences. This copying approach maintains the original gene's regulatory architecture while introducing human coding sequences, providing a reproducible and consistent expression pattern that doesn't depend on complex minigene design variations
3Quantity of substance
If random transgenic integration is used to introduce human genes, then human protein expression is achieved, but the expression level is not normal
Solution Approach 1:
The patent applies local quality by replacing only the exon regions with human sequences while maintaining the native mouse regulatory elements in the introns. This ensures that human protein is expressed at normal physiological levels controlled by the endogenous gene's promoter and enhancers, rather than at abnormal levels due to random integration events
Solution Approach 2:
The patent segments the gene structure to distinguish between coding regions (exons) and regulatory regions (introns). By humanizing only the exons and retaining mouse introns with their native regulatory elements, the system achieves accurate physiological expression levels without the unpredictability of random transgenic integration
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
The exon-humanized mice exhibit normal human protein expression levels and tissue specificity, making them effective models for human disease studies and drug efficacy testing.
Implementation Method 1
the present invention provides a vector expressing a guide RNA for cleaving a genome at a site immediately upstream of a first exon by using CRISPR/Cas9
Implementation Method 2
this homologous recombination is used to knock-in a human gene as cDNA or a minigene into a mouse gene locus
Data Source
AI summary
Provided are a donor vector having an exon-humanized gene in which only exon nucleotide sequences of a mouse gene are replaced with human exon nucleotide sequences, an ES cell in which a mouse endogenous gene is replaced with the donor vector, and a mouse crated by using the ES cell.