CRISPR-Cas9 Humanized Mouse Model for PD-1 Research
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Solution Overview
Problem
Current humanized animal models for studying PD-1 gene-related diseases and immunotherapy have limitations due to significant differences between human and animal physiology, leading to unpredictable drug efficacy and adverse reactions, necessitating the development of a more accurate and effective PD-1 gene-modified humanized mouse model for improved drug screening and research.
Innovation Solution
A method using the CRISPR/Cas9 technique to construct a PD-1 gene-modified humanized mouse model by replacing specific nucleotides of the mouse PD-1 gene with human DNA fragments, ensuring expression of functional human PD-1 protein domains, while retaining mouse PD-1 regions to minimize physiological disruptions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If human genes are introduced into animal models to create humanized models, then the accuracy of studying human diseases is improved, but the physiological differences between humans and animals cause unpredictable drug efficacy and adverse reactions
Solution Approach 1:
The patent applies local quality by selectively humanizing only the PD-1 gene in the mouse model rather than creating a fully humanized model. This targeted approach introduces human-specific PD-1 sequences (including extracellular domain, transmembrane domain, and intracellular domain) into the mouse genome at the specific locus, allowing the model to exhibit human-like PD-1 expression patterns and immune response characteristics while maintaining the overall physiological compatibility of the mouse system. This resolves the contradiction by locally improving human disease study accuracy without fully adopting human physiology that would cause unpredictability.
Solution Approach 2:
The patent segments the PD-1 gene into distinct functional domains (extracellular domain with nucleotides 1-278, transmembrane domain with nucleotides 279-319, and intracellular domain with nucleotides 320-627) and systematically replaces the corresponding mouse sequences with human sequences. This segmented approach allows precise control over which parts of the gene are humanized, enabling the model to capture human-specific immune checkpoint mechanisms while maintaining mouse physiological framework, thus improving study accuracy without sacrificing reliability.
2Manufacturing precision
If CRISPR/Cas9 technology is used to modify genes, then the precision of gene modification is improved, but the complexity of the modification process increases
Solution Approach 1:
The patent applies preliminary action by designing and synthesizing all necessary CRISPR/Cas9 components (guide RNA targeting the mouse PD-1 gene, Cas9 protein, and human PD-1 gene fragments with homology arms) before performing the gene modification. The homology arms (5' homology arm: nucleotides 1-50 matching mouse PD-1, 3' homology arm: nucleotides 628-650 matching mouse PD-1) are pre-prepared to facilitate precise homologous recombination. This preliminary preparation enables the complex CRISPR/Cas9 process to achieve high precision gene modification by ensuring all components are optimized and ready for efficient targeting and integration.
Solution Approach 2:
The patent uses homology arms as intermediaries to mediate between the CRISPR/Cas9 system and the human PD-1 gene integration. The 5' and 3' homology arms serve as bridging sequences that facilitate homologous recombination between the introduced human PD-1 fragment and the endogenous mouse PD-1 locus. This intermediary mechanism simplifies the complex process of precise gene replacement by providing a natural recombination pathway, reducing the need for multiple complex editing steps while maintaining high precision.
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 PD-1 gene-modified humanized mouse model enables more accurate in vivo research and drug screening, enhancing the success rate of early-stage clinical trials and improving the understanding of PD-1/PD-L1 signaling mechanisms, thereby facilitating the development of effective cancer therapies.
Implementation Method 1
The present application provides a method for constructing a PD-1 gene-modified humanized mouse model based on the CRISPR/Cas9 technique
Implementation Method 2
constructing a targeting vector: obtaining a homology arm from genome DNA using PCR, and connecting the homology arm with an hPD-1 fragment to be introduced to the targeting vector
Data Source
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AI summary
Provided is a method for preparing a PD-1 gene-modified humanized animal model. The method utilizes the CRIPSR/Cas9 technique to replace partial fragments of a mouse PD-1 gene with fragments of a human PD-1 gene using homologous recombination by constructing a targeting vector, thereby preparing a gene-modified humanized mouse. This mouse can normally express a PD-1 protein containing the functional domain of the human PD-1 protein, and can be used as an animal model for mechanism research regarding PD-1, PD-L1 and other signals, for screening regulators, and for toxicological research. The method has an important and high application value in studies on functions of the PD-1 gene and in the development of new drugs.