Knockout mice have been essential for gaining proper understanding of gene function, studying human disease mechanisms, and accelerating drug development - playing an important role in biomedical advancements across a variety of research areas. With the development of gene editing technology, knockout (KO) mice become increasingly accessible for researchers worldwide. In this article, we review the basic information on knockout mice research, application examples, and the development process for custom KO mouse models.

 

What is a Knockout Mouse?

A knockout (KO) mouse is a genetically engineered animal model in which certain genes or DNA sequences have been deleted, thus achieving a nonfunctional gene which provides insights on its function. Investigating gene knockout mice characteristics can help scientists have a better understanding of gene functions and advance human disease research.

 

What Are Knockout Mice Used For?

Since humans and mice share about 99% of the same genes across species, knockout mice are an ideal tool for studying human diseases, evaluating new drugs and treatments, as well as discovering gene functions such as unveiling gene’s physical, biochemical and behavioral roles.

Here are some of the typical knockout mouse applications:

1. Discovering Gene Function

The knockout (KO) mouse has been a valuable tool for geneticists to elucidate the biological roles of a genetic allele (and/or encoded protein) in embryonic development, physiological homeostasis, metabolic processes, or development of disease. Due to the similarity of genes between mice and humans, the function of these genes can be analogized to human gene and disease research.

For example, experts from the Chinese Academy of Sciences have studied liver-specific Arid1a knockout mice and found that Arid1a can specifically regulate liver cell reprogramming. If the Arid1a gene is specifically knocked out in hepatocytes, it will inhibit the dedifferentiation of hepatocytes induced by hepatic portal vein injury, leading to defects in the repair of damage.

 

2. Studying Mechanisms of Human Diseases

Using gene editing technology to simulate human diseases in mice is often the best way to study the pathogenesis of diseases in vivo. Knockout (KO) mice serve as valuable animal models for human genetic diseases in which a mutation is found to disable a protein – corresponding KO mice can be used to study the underlying pathophysiology and even develop therapeutics.

For example, the Tnfrsf11b knockout mouse model present typical osteoporosis and arterial calcification phenotypes, which makes it an ideal animal model for studies of human osteoporosis, including: screening and evaluation of therapeutic drugs, developing gene therapy, and studying the interaction mechanism of osteoclasts and osteoblasts, etc.

 

3. Accelerating Drug Development and Efficacy Evaluations

The Knockout (KO) mouse model is an indispensable tool for drug development. Human disease research – such as for developmental disorders and rare disease studies – often use a combination of heterozygous and homozygous KO lines to significantly boost relevant insights into novel diagnostic and therapeutic approaches. For early in vivo evaluation of protein inhibition, pharmaceutical companies often look at the phenotype of a knockout (KO) mouse to understand potential consequences of such a drug development approach.

Take RALY gene for example, in humans RALY is associated with a wide variety of diseases and several genome-wide association studies have connected variants at RALY with cardiometabolic traits, including coronary artery disease and total cholesterol. Mice homozygous for a gene trap allele of Raly are viable, although there are limited studies which have utilized knockout (KO) mouse models for in vivo research of Raly. Cyagen Knockout Catalog Models offers the ready-to-use Raly knockout mouse - C57BL/6J-Ralyem1cyagen – delivered in as fast as 3 months.

 

How Are Knockout Mice Generated?

With the advancements of gene editing technology, genetically modified animal models have become increasingly accessible to scientists. Understanding the process of knockout mouse generation is important for researchers. Cyagen offers comprehensive capabilities and techniques for developing custom knockout mice. Researchers could choose the optimal gene editing technology based on research purpose or request a complimentary project consultation.

 

1. TurboKnockout® Gene Targeting

TurboKnockout® gene targeting method eliminates the unpredictability of germline transmission (GLT) with a super competent embryonic stem cell (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.

 

Highlights of TurboKnockout® Gene Targeting:

No patent risk: Technology of choice for drug development projects

Success rate: 100% guaranteed germline transmission (GLT)

Turnaround time: Founders as fast as 6-8 months

loxP restrictions: No size limitations for your floxed region

Knockout mouse genotyping: Validated genotyping data is available

100% Money-Back Guarantee on TurboKnockout® projects

 

Cyagen | Turboknockout strategy example Figure 1: Turboknockout strategy example

 

2. CRISPR/Cas9-based Gene Knockout

A guide RNA (gRNA) - homologous to an 18-22 nucleotide target sequence in the genome - is used to direct the coexpressed Cas9 nuclease to the target site. Hybridization of the gRNA localizes Cas9, which then cuts the target site in the genome, generating double-strand breaks (DSBs). Cellular repair of DSBs by the nonhomologous end-joining pathway (NHEJ) generates stochastic errors at the cut site. This usually results in small deletions, or - more rarely - insertions and base substitutions. When these mutations disrupt a protein-coding region (e.g. a deletion causing a frameshift), this will result in a functional gene knockout.

Cyagen | Figure 2: CRISPR knockout strategy example

Figure 2: CRISPR knockout strategy example

 

Comparison of Knockout Model Generation Methods

 

TurboKnockout®

CRISPR/Cas9 Gene Editing

Traditional Gene Targeting

Turnaround time

6-8 months

5-7 months

10-14 months

Approach

Homologous recombination in ESC by Cyagen’s TurboKnockout® technology

CRISPR/Cas9 nuclease mediated gene targeting by pronuclear injection

Homologous recombination in ESC by traditional technology

Self-deleting selection cassette

Yes

N/A

No

No need to breed to Flp deleter mice

Requires breeding to Flp deleter mice

Screening method

PCR + Southern blot

PCR + Sequencing

PCR + Southern blot

Patent Risks

N/A

High-risk

N/A

 

Cyagen’s Knockout Mice Resources

Cyagen’s gene editing services are unparalleled in delivering research-ready knockout mouse models with a guaranteed genotype. Below we have collected all our knockout mouse resources:

 

Animal Model Supporting Services

In addition to animal model generation, Cyagen has established a range of supporting services, such as breeding, embryo/sperm cryopreservation, histology, and more. Outsourcing these downstream services to Cyagen is a more cost-effective way to conduct your research. All these services are performed by experienced specialists following standardized procedures, so we guarantee to deliver high quality services and data with unbeatable price and turnaround.

 

Cyagen Knockout Catalog Models

The Cyagen Knockout Catalog Models repository provides a searchable listing of over 10k custom KO and cKO/floxed strains from which live mice are available to researchers in as few as three (3) months. Our repository is constantly growing, so feel free to inquire about the status of any specific gene(s) not shown online for more information about pending availability.

 

Contact Our Model Generation Experts

From strategy design through to delivery of research-ready custom models, Cyagen offers complete outsourcing for all your animal model needs. We can create any custom mouse or rat model with guaranteed genotype validation, even offering phenotypic characterization and other downstream services required to establish a mouse model for your research project.

Contact us with your project details to request your complimentary model generation project consultation, strategy, and quote.

 

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