Knockout Mice

This model is engineered to carry an inoperative gene. Usually, inactivation of the gene is achieved by the deletion (partial or total) of its sequence, and it is nonfunctional in the entire animal.

These knockout mice allow the deletion of a gene in a tissue- and/or time-specific manner. Often, the conditional KO mouse is achieved through the Cre-lox system. Instead of deleting the critical sequence, it is flanked by loxP sites (termed a floxed sequence). Cre recombinase deletes the sequence between two loxP sites. An inducible or tissue-specific Cre is used to knockout gene function in only that tissue.

RNA interference can reduce the expression of a target gene, without deleting it. Typically this is combined with conditional and/or inducible approaches to reduce expression of the target only under certain conditions in adult animals.

Knockout mice are genetically modified mice that have one or more genes intentionally removed or "knocked out" in order to study the function of that gene in relation to a particular disease or condition.

Knockout mice are important in biomedical research because they allow scientists to study the function of specific genes and their roles in diseases or conditions. By removing a particular gene, researchers can observe the effects on the mouse's physiology and behavior and gain insight into how the absence of that gene may affect humans.

Knockout mice are created using a process called gene targeting, which involves replacing a specific gene with a non-functional copy. This can be done using techniques such as homologous recombination or CRISPR/Cas9* gene editing.

Knockout mice have been used in cancer, cardiovascular disease, neurodegenerative disorder, and immunology studies. Some examples include studying the role of tumor suppressor genes in cancer, investigating the molecular mechanisms of Alzheimer's disease, and exploring the immune response to viral infections.

Knockout mice allow researchers to study the specific roles of genes in vivo, which can provide valuable insights into the mechanisms of disease and potential therapeutic targets. They also allow for the testing of new drugs and treatments in a controlled and standardized laboratory setting.

To ensure the validity and reproducibility of their results, researchers use rigorous experimental designs and statistical analyses, as well as carefully controlled conditions to minimize variability. They also publish their findings in peer-reviewed journals and share their data with the scientific community for further scrutiny and replication. It is also essential to check that the knockout model leads to total inactivation of the targeted gene. It should also be borne in mind that the results obtained have only been achieved in a single experimental and genetic context.

Knockout mice are particularly useful in oncology research because they allow researchers to study the effects of specific genes on tumor growth and development. By creating mice that are missing a particular gene associated with cancer, researchers can gain insight into the role of that gene in the development and progression of tumors.

Knockout mice are used in neuroscience research to investigate the effects of gene mutations on brain development, function, and behavior. Researchers can study the behavioral and physiological effects of a particular gene's absence by comparing knockout mice to normal, wild-type mice.

The gene knockout method is the simplest approach to reveal fundamental gene functions. Conditional knockout/inductible mice, inducible knockout mice, and knockdown mice can avoid the risk of a lethal phenotype associated with gene inactivation at earlier stages in development and allow for an analysis of gene inactivation effects on adult animals. Furthermore, as 65% of protein coding genes are likely pleiotropic, a conditional knockout mouse simplifies phenotyping analysis by focusing on a specific cell type.

Disrupting genes can sometimes result in compensation by other members of a multigene family. In these cases, multiple knockouts may be needed to obtain phenotypical effects (e.g., Hox genes). Moreover, it is possible that gene knockouts may fail to produce observable phenotypes in knockout mice, or may produce different characteristics from those observed in humans. Mouse background choice is an important consideration for better mimicking human phenotypes – ask our experts for further advice.

Depending on your study and objectives, knockout mice can be obtained through:

• Genome-editing nucleases (e.g., CRISPR knockout)
  • One-cell embryo direct injection
  • ES cell modification

For further information on knockout mice, please see the International Knockout Mouse Consortium (IKMC) website.

The Science of Controlling CRISPR
With technologies like CRISPR gene editing accelerating at a rapid pace, what was once thought to be impossible to achieve with mice may become reality sooner than we ever imagined. The challenge will be to fully understand and control these new mouse model creation tools. For example: how does one limit off-target effects? What do we know about circular permutation, or inhibiting CRISPR with anti-CRISPRs? Read the full story

The CRISPR Potential in Accelerating Drug Development
CRISPR's comparative ease of use is expected to contribute to the development of more multifaceted cellular assays with improved predictability for drug therapies. Some of the benefits include reduced attrition rates of compounds and improved target validation. Read the full story

We refresh every five generations to avoid creation of a substrain.

Refreshing breeder colonies for knockout mice is an important requirement and should be performed on regular basis every 5 – 10 generations of breeding. This will help to mitigate the risk posed by genetic drift and the occurrence of spontaneous mutations that may impact mouse phenotypes and experimental data bias.

Refreshing can be done in two ways. The first method is to revitalize cryostock. Cryopreservation should be done when we can be confident that the line line has not already drifted.

The second way would be to breed a few generations (three generations are recommended) with wild-type animals purchased from genetically controlled colonies. Remember to first breed transgenic females with WT males to refresh the Y chromosome.

Use of an appropriate wild-type and/or HE control will tell help you evaluate where exactly the change in phenotype came from with your knockout mice. If the phenotype is induced by the mutant target gene then the controls should not show the phenotype.

Always backcross the first KO mouse females with wild-type males to refresh/exchange the Y chromosome. Then in subsequent breedings, use transgenic males obtained from the first breeding round to mate with wild-type females.

Theoretically speaking, brother/sister mating is really the only way to propagate an inbred strain, especially for larger colonies. For smaller colonies, it is possible to do it differently, but only if the colony is regularly refreshed following a well-designed strategy.

Depending on the test used, with STR markers, certain testing organizations advise the use of at least three individuals. Most of the time, testing 3 – 5 individuals is enough to get informative indication on genetic background purity.