Humanized mice can be developed through a variety of genetic modification techniques, wherein human genes may directly replace endogenous mouse genes or be inserted into a safe locus, such as Rosa26. Such humanized mouse models have been widely used in studies of human gene function, immune-oncology therapeutics, infectious disease pathology, and preclinical drug evaluation in the field of biomedicine. 

For example, through the humanization of mouse genes related to immune checkpoints (such as CTLA4 and PD1, etc.), researchers can develop humanized mice that can interact with anti-CTLA4 and PD1 humanized antibodies. This approach enables mice to be used as tools for pre-clinical screening and efficacy evaluation for new drugs, given their improved ability to predict human responses to treatments.

With the rapid advancement of humanized antibody production, the use of genetic modification to develop mice that produce human antibodies has become an important facet of antibody drug development research. A humanized mouse expressing human antibody can be obtained through multiple rounds of gene targeting, respectively replacing the homologous mouse region with the human antibody heavy chain/light chain variable region. After being stimulated by the corresponding antigen, the humanized mouse can produce an antibody containing a humanized variable region portion against the antigen along with the constant region of a mouse antibody. Studies have shown that human antibodies obtained through humanized mouse screening have a more effective affinity and activity  compared with human antibodies obtained by in vitro DNA recombination methods.

 

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Strategies for Developing Humanized Mouse Models

At present, humanized mouse models are mainly constructed using the following genetic modification methods:

1) Introduce desired human transgene sequence into mice by pronuclear injection (PNI), wherein the human sequence replaces the endogenous gene of interest via random integration – resulting in a transgenic (TG) mouse that expresses human genes;

2) Use nuclease-induced genome editing technology to construct a site-specific humanized mouse model – Cyagen’s CRISPR-based Rapid Knockout uses a proprietary pronuclear injection (PNI) cocktail and PNI technique that significantly enhance targeting efficiency;

3) Use targeted mouse embryonic stem cell (ESC) line for traditional gene targeting to achieve the purpose of replacing mouse genes or inserting human genes via homologous recombination (HR);

4) Cyagen’s TurboKnockout® gene targeting service, a proprietary optimization of traditional ESC-mediated targeting techniques, enables more efficient and stable gene integration for the development of humanized mouse models.

 

Although transgenic (TG) models have played a significant role in the research discovery process for diseases, they do have a limited ability to accurately recapitulate some aspects of human disease processes. Towards this end, more complicated projects may necessitate the use of an alternative gene modification technique to achieve an improved humanization model. Knock-in (KI) models offer more accurate genetic targeting capabilities that enable investigation of specific genes’ roles in human diseases. Compared with TG models, KI models have the following 3 advantages:

  1. Insertion is non-random, and genetic impacts are controlled at the lowest level;
  2. Without multiple copies, models more closely recapitulate the pathological features of human diseases;
  3. Most of the endogenous genome sequence is retained so that the original regulatory elements can retain control.

 

While either nuclease-induced genome editing or ESC-mediated gene targeting may be used to develop humanized KI mouse models, each method offers its own benefits for model development. The TurboKnockout® gene targeting service brings together the advantages of each method - providing complex gene modeling in C57BL/6 or BALB/c mouse models, including large-fragment knockins (LFKIs) up to 200 kb. Compared with CRISPR/Cas9-based gene engineering, TurboKnockout® is free of patent disputes and is the technology of choice for drug development projects - providing conditional knockout, reporter knockin, and humanization mouse models in as fast as 6 months.

 

TurboKnockout® Gene Targeting Mice Service

Our proprietary TurboKnockout® gene targeting method eliminates the unpredictability of germline transmission (GLT) with our super competent ESC line that generates 100% ESC-derived founder mice rather than chimeras. Additionally, self-removing selection cassette circumvents the need to breed to Flp deleter mice. For conditional knockouts, it may be possible to breed founders directly to your tissue-specific Cre mice. These innovations eliminate at least two generations of breeding, shortening production time by 4-6 months as compared to the industry standard.

 

About Cyagen

In under 15 years since its founding, Cyagen has become a leading provider of custom mouse and rat models – delivering over 50,400 models to researchers worldwide and receiving over 3,600 peer-reviewed citations. Cyagen’s top-quality services are backed by our industry-best 100% money-back guarantee. We are known to provide the industry’s best price, quality and turnaround time on all our model generation services – see for yourself from our customer testimonials:

 

"We generated a mouse knockout model using Cyagen’s TurboKnockout® technology. Cyagen’s team was on time with milestones, provided constant updates on the project and delivered animals as promised. It was such a pleasure working with Cyagen that we decided to outsource the production of other animal models to them."

— Maximiliano D’Angelo, Sanford Burnham Prebys Medical Discovery Institute

 

Cyagen provides free consultations, strategy designs, and quotes from our team of gene modification experts – contact us today to see how we can support your research!

 

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