In therapeutic applications, monoclonal antibodies (mAbs) provide highly precise targeting of pathological cells and reduced side effects in treating disease, which has led to their wide use in clinical research. Experimental mice are widely used models for antibody drug development, with their advantages including low immune tolerance to human antigens and easy operation. However, human rejection caused by mouse-derived antibodies seriously affects the safety and efficacy of such antibody therapy. Therefore, the humanization of mouse-derived antibodies has become the main focus for major pharmaceutical companies to tackle key limitations of antibody therapy research.
With the rapid development of human antibody production, using gene editing technology to construct humanized mice with human antibodies has become an important subject in antibody drug discovery. However, due to the large genomic region of human antibody Ig genes, it makes the construction of humanized mice that express the human antibody tremendously challenging. Accordingly, it remains very challenging to achieve large-fragment human antibody gene insertions in animal models, especially for a gene that is greater than 1 Mb.
Speeding Up Antibody Discovery with Cyagen’s TurboKnockout® , RMCE, and BAC Technology
Cyagen is able to generate large fragment knock-in (LFKI) humanized mouse models that express human antibody genes, by using TurboKnockout® RMCE and BAC technology.
Using data from thousands of knock-in mouse model projects completed by our team, we have collected new information demonstrating how our gene editing technologies push the boundaries of modifying large genomic regions.
Based on TurboKnockout® (ES) cell targeting techniques, we have used RMCE-based techniques (with BAC fusion and 3 targeting rounds) to achieve large-fragment knockin (LFKI) humanization and genetic modifications for regions up to 300 kb!
Importantly, the project timeline can be significantly shortened through multi-step BAC reorganization at the ES cell level, which can greatly salve your same and project costs, speeding up antibody discovery.
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1) What is TurboKnockout® Technology?
The TurboKnockout® gene targeting service by Cyagen is based on traditional embryonic stem (ES) cell-mediated targeting techniques and can be used for complex gene modeling projects to provide C57BL/6 or BALB/c mouse models with accurate genetic modification of large genomic regions. This proprietary gene targeting method eliminates at least two generations of breeding, shortening production time by 4-6 months as compared to the industry standard.
2) What is Recombinase-Mediated Cassette Exchange (RMCE) and BAC?
Recombinase-mediated cassette exchange (RMCE) implements site-specific recombination to achieve systematic, repeated modification of higher eukaryotic genomes. The recombinase-mediated cassette exchange (RMCE) approach remains a powerful tool for the insertion of large gene fragments, and may be used to efficiently generate conditional, reporter, and transgenic mouse models. RMCE provides highly efficient transfer of genes of interest (GOIs) and is readily adapted to generate humanized mouse models – reproducing the human expression patterns in mice.
Bacterial artificial chromosome (BAC) is a low-copy vector that can hold more than 300 kb of foreign DNA. Through BAC recombination, larger genes and regulatory sequences can be introduced - which is closer to the expression pattern of endogenous genes.
The establishment of a mouse model expressing a human antibody provides a reliable and irreplaceable platform for the development of therapeutic antibody drugs. In this White Paper, our experts review the whole process of antibody drug development and analyze the various strategies used in generating human antibody mouse models.
- ● How are Therapeutic Antibodies Developed?
- ● Important Considerations in the Humanization of Antibodies
- ● Human Antibody Discovery Using In-vivo Mouse Models
- ● Leveraging Humanized Mice for Human Antibody Discovery
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Humanized Mice Generation Process
Advantages of Cyagen Human Antibody Mouse Models Platform
1) Super competent ES cell line generates 100% ESC-derived founders, avoiding the chimera phase;
2) Self-removing Neo selection cassette that circumvents the need to breed to Flp deleter mice.
In addition, multi-step BAC reorganization by TurboKnockout® at the ES cell level can shorten the production cycle.