Mice are commonly used as a mammalian model for studying of human diseases. They are small, cost-effective, reproduce quickly and easy to handle. They are both biologically and genetically similar to humans, and most importantly, mouse genes can be easily manipulated.

Why do we Study Knockout Mouse?

A knockout mouse is a genetically modified mouse in which a specific gene is inactivated or “knocked out” by introducing an artificial DNA sequence to replace or disrupt the existing gene. Knocking out the activity of the gene provides information about the function of the gene. Knockout mice are useful in studying and modelling different kinds of human diseases, including cancer, diabetes, obesity, cardiovascular disease, neurogenerative disease, and aging.

How is a Knockout Mouse Made? 

There are multiple strategies to create a knockout mouse model, such as embryonic stem (ES) cells and clustered regularly interspaced short palindromic repeats (CRISPR), all with the aim to prevent the translation of the gene’s full length wild-type sequence. 

Gene Targeting Using Embryonic Stem Cells

Homologous recombination in ES cells is a robust method for specific gene targeting of large genomic regions. This is accomplished by inserting a foreign DNA sequence directly into the specific gene of interest using a homologous recombination vector. The foreign DNA sequence is inactive, and it bears a “reporter gene” to help in screening for cells where the insertion was successfully introduced. The modified ES cells are injected into blastocyst to form a chimeric embryo which develops into a chimeric mouse. Chimeric mice are then bred with wild-type mice to ensure germline transmission of the modified genetic material to its offspring.

CRISPR/Cas9 Technology

The CRISPR genome editing system is a RNA-guided endonucleases system to target small DNA sequences of interest. In the presence of guide RNA (gRNA) and Cas9 endonuclease, the gRNA directs the Cas9 endonucleases to cleave the DNA at a specific location, creating indels in the gene, inducing a frameshift mutation, generating a constitutive knockout. gRNA and Cas9 are injected into embryos, followed by implantation into foster mothers to generate founders. Founders are then screened and bred to obtain F1 mice bearing the knockout allele.

The CRISPR/Cas 9 technology is a simple and rapid method for generating and obtaining knockout mouse models. It is much faster than the conventional method using gene targeting in ES cells, which involves many months of targeting vector construction, ES cells selection, and validation of targeted clones and achieving germline transmission from at least one clone. 

Limitations in Knockout Mouse

There are several limitations to knockout mice. About 15 percent of gene knockouts are developmentally lethal, which means that the genetically altered embryos cannot grow into adult mice.  In some cases, the genes play different roles in embryological stages and adulthood. Furthermore, the loss of the gene activity during development may mask the role of the gene in the adult state. Some of these limitations maybe overcome through the use of conditional knockout, where the target gene is inactivated in a specific tissue or an inducible basis to allow the mouse to develop to adult sate before inactivating the gene of interest.  

The Knockout All Project – A Comprehensive Mouse Resource

In 2019, GemPharmatech launched the Knockout All Project (KOAP) with the ambition to generate knockout or conditional knockout mouse strains for all protein-coding genes in the mouse genome within 5 years. KOAP creates mouse models via a high throughput CRISPR based gene editing strategy, with the capacity of creating around 6000 mutant strains each year. Nearly 10,000+ genes have been targeted so far, providing over 20, 000 available knockout and conditional knockout mouse models for a wide array of research fields including oncology, autoimmunity, metabolism, and neurodegeneration.  

Production timeline of KOAP. It is estimated that by 2023, the whole KOAP will be completed.

Transgenic mice expressing Cre recombinase are required for the generation of conditional knockout mouse.  GemPharmatech has generated 199 mouse strains expressing Cre recombinase in specific tissues, such as immune system, nervous system, bone, muscle and adipocytes, gastrointestinal tract, cardiovascular system, reproductive system and so on. The KOAP mice are created in the C57BL/6 background strain. Likewise, the Cre lines are also created in C57BL/6 background strain, making it feasible to breed with flox strains created in KOAP to produce conditional knockout mice. Codon-Improved Cre Recombinase (iCre), demonstrating to express at a higher level in mouse, was employed in most GemPharmatech Cre lines to allow high efficacy excision.

Statistics of Cre lines. 165 of 199 Cre strains have been recorded in MGI and 34 strains are novel. CreER versus constitutive, 52 versus 147; H11/R26 KI versus 3′ endogenous KI alleles, 49 versus 123.

Ready-to-Use Knockout Models

As the leader in genetically engineered mouse models, through KOAP, GemPharmatech envisions to create the largest collection of CRISPR mouse strains, providing researchers valuable mouse resources to study human diseases. 

We highly recommend scientists and researchers to check out the huge library of mouse resource. If your desired gene of interest is not available, fret not, we can create one in as little as 3-6 months.

Why wait? Contact us now!

Reference : Ju, C., Liang, J., Zhang, M. et al. The mouse resource at National Resource Center for Mutant Mice. Mamm Genome 33, 143–156 (2022). https://doi.org/10.1007/s00335-021-09940-x

Tags: conditional knockout, CRISPR, ES technology, GEMMs, Knockout All Project, knockout mouse, KOAP, Mouse models, transgenic mouse model