Since the introduction of knockout animal models, advancements in genetic engineering technology have led to a revolution in research using genetically edited models (GEMs). In the knockout process – usually a conventional or conditional knockout mouse model – an artificial DNA piece is introduced to disable or replace the gene.
The knockout model is important to scientific studies due to its role in finding numerous hidden details like why and how a certain gene behaves as it does. Moreover, these details also help in realizing why the certain gene is impaired and what changes can lead to restoring its natural behaviour.
Significance of Knockout Mouse Models
Mouse models have been holding the foreground in a large percentage of studies devoted to understanding DNA sequences and gene function research. In addition to their small size, and genetically similar make-up to humans, the specific method required in the knockout technique can easily be applied to mice, compared to other laboratory animals.
What is Conventional Knockout Mouse?
Conventional knockouts (KOs), wherein a gene is disabled throughout an entire organism, were among the first genetically modified animal models to be created. A KO model helps in uncovering the role of the gene in every area of the organism. This KO model gives a broader view to the researchers about how gene expression can influence even the small and subtle processes in the organism and what role it plays in the development of various diseases.
What is Conditional Knockout Mouse?
In a conditional knockout (cKO) mouse, a specific gene is eliminated or disabled from a certain tissue but not from the entire organism. Conditional knockout (cKO) models can be of two types, namely tissue-specific and inducible knockouts.
- Tissue-specific: Tissue-specific knockout is when a gene of interest inactivates in a certain tissue in a specific cell type, keeping other cell types and tissues unmodified and gene expression functional.
- Inducible: Inducible knockout can temporarily suppress the gene of interest at a given time point in the fetal, postnatal, or adult animal.
For example, in a conditional knockout mouse to study the effect of diseases in the liver, the scientist might eliminate a particular gene only from a specific organ (liver), and the results can be more accurate regarding genetic therapies or drugs they develop, which can be of such great help in the long run.
Some cells are essential in embryonic development. If the conventional gene knockout can affect all cell types unlike the conditional knockout process, the mice may not survive gestation or die soon after birth due to embryonic lethality.
With conventional knockout, the gene of interest will be disabled in all the tissues and at all the time, whereas in conditional knockouts, the gene is active except in the specified tissues (tissue-specific cKO) or at the desired time point (inducible cKO).
Thus, since the gene is always inactivated in conventional KO models, it is prohibitive to understanding the gene expression within the mice at different stages of development, or how activation and deactivation of the desired gene may influence related pathologies. With conditional knockout, the researcher knocks out genes at a particular stage in development and can study how a disabled gene in one tissue affects the same (unmodified) gene in other tissues.
Why is Conditional Knockout Mouse a Preferred Choice?
It is important to understand that conditional knockouts have more advantages and capabilities than conventional knockouts. Conditional knockout (cKO) models are often referred to as ‘floxed’ models due to the use of Cre-lox recombination system in their development.
With the advantages of simple operation and high recombination rate, the Cre-Lox system has now become a powerful tool for genetic manipulation across in vivo and in vitro models – used for the study of gene function and modelling of human disease.
The Cre-Lox system is often used to achieve custom spatiotemporal operations on specific genes and greatly improves the efficiency in generating conditional mouse. Mating a loxP mouse with a specific Cre line available in repositories makes the gene inactivation possible at a specific time point and/or in a certain tissue type in conditional knockouts.
Both the conventional and conditional knockout mice were first made using traditional gene editing, which is mediated by embryonic stem (ES) cells. This method involves manipulating the gene of interest in embryonic stem (ES) cells, after which, ES cell clones are screened for proper targeting before injection into blastocytes to produce live mice with the modified gene.
Now, advanced technologies like CRISPR can be employed to directly inject zygotes, bypassing the time-consuming approach of traditional embryonic stem cell-mediated model generation. Scientists are more likely able to achieve a knockout sooner, and in some cases, homozygous knockouts are found right from the beginning.
Prospect: Cutting-Edge Gene Editing with Conditional Knockouts
After the development of CRISPR and other advanced genetic engineering technologies for achieving conditional knockout (cKO) models, these techniques have led to cKO models’ increasing usage compared to constitutive knockouts. The newer techniques maintain efficiency, simplicity, and highly reduce the time needed for obtaining the modified target genes. Additionally, these strategies are often augmented with the use of bioinformatics tools to identify appropriate RNA sequences for vector construction.
In conclusion, both conventional (constitutive) and conditional knockout (cKO) models are useful research tools, but cKO models provide unique advantages. The additional flexibility provided by conditional knockouts (cKOs), especially with new technologies such as Cre-Lox providing spatiotemporal control of gene expression, has led researchers to increasingly implement conditional knockout methods – rather than the older conventional knockout mouse models.
Cyagen is a global leading provider of custom mouse and rat models for scientific research institutions and the pharmaceutical industry. Their cutting-edge mouse model generation technologies enable them to offer a broad portfolio of custom genetically engineered models, including conditional knockout mouse models, one of their main custom models. With Cyagen’s unique genotype guarantee available for custom conditional knockout (cKO) mouse model services, you can rest assured and be ready to go with your experiments.