Chimeric c-KIT Mouse Model for Human Inhibitor Testing
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Solution Overview
Problem
Existing methods for introducing human c-KIT protein into animal models face challenges such as incorrect expression, localization, and posttranslational modifications, leading to impaired signaling and functional impairment of chimeric proteins, especially when human and mouse c-KIT proteins are used in combination.
Innovation Solution
Generation of transgenic mice expressing a chimeric c-KIT protein with a mouse signal peptide, human extracellular domain, and mouse transmembrane and intracellular domains, integrated into the mouse genome at a specific locus, enabling correct expression and binding to mouse stem cell factor (SCF).
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If human c-KIT protein is introduced into animal models, then the ability to test anti-human c-KIT inhibitors is improved, but the protein expression, localization, and posttranslational modifications are impaired
Solution Approach 1:
The patent applies local quality by creating a chimeric protein where only specific domains are humanized (extracellular domain for inhibitor binding) while other domains remain murine (transmembrane and intracellular domains for proper cellular processing and signaling). This localized humanization resolves the contradiction by maintaining compatibility with the mouse cellular environment while preserving the ability to bind human-specific inhibitors.
Solution Approach 2:
The chimeric c-KIT protein serves as a composite structure combining human and mouse protein domains. The human extracellular domain (amino acids 26-524) is fused with the mouse transmembrane domain (amino acids 525-546) and intracellular domain (amino acids 547-1044), creating a hybrid protein that exhibits properties of both parent proteins: ability to bind human inhibitors and ability to function properly in mouse cells.
2Reliability
If chimeric c-KIT protein with human extracellular domain and mouse transmembrane/intracellular domains is constructed, then binding to mouse SCF and cell proliferation signaling is improved, but the sequence similarity between human and mouse c-KIT proteins creates uncertainty about functional compatibility
Solution Approach 1:
The c-KIT protein is segmented into three functional domains: extracellular domain (human, for ligand binding), transmembrane domain (mouse, for membrane anchoring and conformational transmission), and intracellular domain (mouse, for signal transduction). This segmentation allows each domain to be optimized for its specific function while maintaining overall protein integrity.
Solution Approach 2:
The chimeric c-KIT protein acts as an intermediary that bridges human inhibitor binding requirements with mouse cellular processing requirements. The human extracellular domain mediates inhibitor binding while the mouse transmembrane and intracellular domains mediate proper folding, trafficking, and signaling in mouse cells.
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 chimeric c-KIT protein effectively binds to mouse SCF, mediating cell proliferation and survival, providing a reliable platform for testing anti-human c-KIT inhibitors.
Implementation Method 1
C-KIT binds to stem cell factor (SCF) and forms a dimer that activates its intrinsic tyrosine kinase activity, which in turn phosphorylates and activates signal transduction molecules
Implementation Method 2
Protein processing of c-KIT includes N-linked glycosylation and phosphorylation on both tyrosine and serine residues
Implementation Method 3
Protein processing of c-KIT includes N-linked glycosylation and phosphorylation on both tyrosine and serine residues
Data Source
AI summary
The present disclosure describes methods of generating a transgenic cell or mouse that expresses a chimeric c-KIT protein capable of expression on mouse cells and binding to mouse stem cell factor (SCF) which can be inhibited by anti-human c-KIT inhibitors. The transgenic cells and mice can be used to test candidate anti-human c-KIT inhibitors.