Knockout Mouse Catalog | Cyagen APAC

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 difficult to achieve large-fragment human antibody gene insert in animal models, especially for gene that is greater than 1 Mb.


How to Overcome the Challenges of Generating Humanized Mouse Models for Human Antibody Development

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.

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 eliminate at least two generations of breeding, shortening production time by 4-6 months as compared to the industry standard.

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.

TurboKnockout®, RMCE, and BAC: Speeding Up Antibody Discovery

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.

CRISPR/Cas9 Versus TurboKnockout®



CRISPR/Cas9 Gene Editing

Turnaround time

6-8 months

5-7 months


Homologous recombination in ESC by Cyagen’s TurboKnockout® technology

CRISPR/Cas9 nuclease mediated gene targeting by pronuclear injection

KI fragment size

Up to 300 kb*

Usually less than 5 kb

Self-deleting selection cassette

No need to breed to Flp deleter mice


Screening method

PCR + Southern blot

PCR + Sequencing

Patent Risks



Off-target Effect




Limitations of CRISPR/Cas9-mediated Gene Editing

In terms of drug development, CRISPR technology is the most widely used technology in generating gene editing animal models. Although CRISPR is known for advantages such as high efficiency, rapid turnaround, convenient process, low cost, and successful application to different species; the disadvantages include the unpredictable and uncontrollable off-target risks, fragment size limitations, and ongoing patent disputes – often making it unsuitable for complex genetic modification projects.

With the challenges faced by CRISPR, ES cell-mediated targeting technology has remained an indispensable tool for pharmaceutical companies to generate humanized mouse models. Notable disadvantages of standard ES cell-mediated approaches include inefficiency, time-consuming process, labor-intensive, costly, and species limitations.

Cyagen’s innovative TurboKnockout® technology is an advanced version of ES cell-mediated gene targeting, which has shortened the construction turnaround time from one year to as few as six months – on par with CRISPR - and simultaneously reduced the overall project costs. Compared to CRISPR/Cas9, TurboKnockout® provides accurate genetic changes without off-target effects and can perform complex genetic modifications – exceeding the industry standards.


TurboKnockout Advantages

  • Freedom to Operate: Technology of choice for drug development projects

Compared with CRISPR/Cas9 techniques, TurboKnockout® is free of patent disputes and is the technique of choice for new drug development projects. No risk of patent infringement.

  • Success Rate: 100% guaranteed germline transmission (GLT)

TurboKnockout® is based on traditional ES cell targeting techniques; it can be used for complex gene knockouts for mice, providing models with accurate genetic modification, 100% germ line transmission, and no off-target effects.

  • Shorter Turnaround: Founders as fast as 6-8 months

Reduced timeline due to several innovations that eliminate two generations of breeding, and more:

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.

  • Large Fragment Knockin: Insert fragments up to 300 kb

Limited by the capacity of DNA cloning vectors, foreign gene fragments integrated into genetically modified animals are usually less than 30kb. At this size limitation, key elements in regulating gene activity are often lost. BAC can accommodate foreign DNA of more than 300 kb. By introducing larger genes and regulatory sequences through BAC modification, this allows greater control over the expression pattern of genes.

  • Flexible Floxing: No size limitations for your floxed region

With the advantages of simple operation and high recombination rate, the Cre-Lox system has become a powerful tool for genetic manipulation across in vivo and in vitro models. The Cre-Lox system enables gene expression with exceptional spatial (expresses a gene in specific cells, tissues, or entire organisms) and temporal (at specific time points through the application of the Cre-Lox system) control.

  • 100% Money-Back Guarantee 


Related Reading

>> CRISPR/Cas9 Versus ES Cell Targeting:Which is the Optimal Solution?


White Paper on Human Antibody Discovery Research

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.

Outline of Contents

● 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

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.

<< Download Free White Paper on Human Antibody Discovery Now!

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