The Difference: Knockout vs. Conditional Knockout Mice?

The Difference: Knockout vs. Conditional Knockout Mice?

The Difference: Knockout vs. Conditional Knockout Mice?


Mice are frequently employed as model organisms to study human diseases and genetic disorders. One of the most powerful tools in mouse genetics is the generation of knockout (KO) and conditional knockout (cKO) mice. These mice have been widely utilised to understand gene function, disease mechanisms, and drug development. Nonetheless, there is often uncertainty between KO and cKO mice. This article aims to clarify the crucial distinctions between these two varieties of mouse models.

What are Knockout Mice?

Knockout mice are genetically modified mice in which a specific gene of interest has been “knocked out” or deleted. This is achieved using advanced genetic engineering techniques, such as homologous recombination or CRISPR/Cas9 genome editing, which enable precise targeting of specific genes in mouse embryonic stem cells.

Once the targeted gene is deleted or disrupted, the cells are then used to create a mouse embryo, which is implanted into a surrogate mother for gestation. The resulting KO mouse completely lacks the function of the targeted gene in all cell types throughout development, allowing researchers to study the gene’s function and the effects of its absence on the organism.

KO mice can be homozygous (both copies of the gene are deleted) or heterozygous (only one copy of the gene is deleted). The use of homozygous versus heterozygous KO depends on the research question being addressed. Homozygous KO mice are useful for studying the complete loss of function of a gene, while heterozygous KO mice are useful for studying the effects of a partial loss of function or haploinsufficiency.

What are Conditional Knockout Mice?

Conditional knockout mice are generated by selectively deleting a specific gene in a particular tissue or at a specific time during development. This is typically accomplished using the Cre-loxP system which allows for precise control of gene expression.

In this system, the targeted gene is flanked by two loxP sites which act as a switch that can turn the gene on or off depending on the presence or absence of a specific enzyme called Cre recombinase.

Cre recombinase is expressed under the control of a tissue-specific promoter or an inducible promoter, allowing for spatial or temporal control of gene expression. When Cre recombinase is expressed, it deletes the gene of interest between the two loxP sites. This results in the deletion of the gene only in specific tissues or at specific times, which allows researchers to study the effects of gene deletion in specific tissues or at specific developmental stages.

Similar to the KO mouse, to generate a cKO mouse, the targeted gene is modified to contain loxP sites, and the modified gene is introduced into embryonic stem cells. The modified cells are then used to create a mouse embryo, which is implanted into a surrogate mother for gestation. The resulting mouse will carry the modified gene in every cell of its body, but the gene will be functional until Cre recombinase is introduced.

To create a tissue-specific/time-dependent cKO mouse model, a mouse carrying the conditional allele (loxP-flanked) of the target gene is typically crossed with a transgenic mouse line expressing Cre recombinase under the control of a tissue-specific promoter or an inducer. When the Cre recombinase is expressed in a particular tissue or in the presence of the inducer, it catalyzes recombination between the loxP sites, resulting in the deletion of the targeted gene only in the tissue or when the inducer is present.

This method of generating cKO mice allows researchers to investigate the role of specific genes in a particular tissue or organ without affecting the gene’s function in other tissues. It also enables the investigation of the temporal regulation of gene expression during development or disease progression.

Visual explanation on the different types of knockout mice used in research labs
Schematic diagram of constitutive, conditional, and inducible knockout mouse models.
Credit: Delgado Caceres M, Pfeifer CG, Docheva D. doi:10.1089/scd.2018.0121 reproduced under the Creative Commons license

What are the key differences between knockout and conditional knockout mice?

1. The specificity of gene deletion between KO and cKO mice is different

KO mice lack the function of the targeted gene in all tissues and at all times. In contrast, cKO mice allow researchers to study the effects of gene deletion in specific tissues or at specific developmental stages. This allows for a better understanding of gene function and disease mechanisms.

2. Method of gene deletion between KO and cKO mice is different.

KO mice are typically generated by deleting a gene from the mouse genome using gene targeting techniques such as homologous recombination or CRISPR/Cas9 genome editing. In contrast, cKO mice are generated using the Cre-loxP system, which allows for precise control of gene expression in specific tissues or at specific developmental stages.

3. The intended use of KO & cKO mouse models differs.

KO mice are typically used to investigate the role of a gene in a specific biological process or disease state by studying the phenotype resulting from the complete loss of gene function. In contrast, cKO mice are used to investigate the tissue-specific or temporal roles of genes in biological processes or disease states, with the aim of understanding the precise mechanisms underlying these processes. Additionally, the cKO mice can allow the study of embryonic lethal conditions and mimic late-onset pathology which is not possible with KO mice.

4. The phenotypic effects of the gene inactivation may differ between KO and cKO mice, due to compensatory mechanisms that can occur in KO mice.

In cKO mice, the targeted gene is inactivated in a specific tissue, allowing compensatory mechanisms to occur in other tissues, whereas in KO mice, these compensatory mechanisms are absent.

In summary, KO and cKO mice are powerful tools in mouse genetics. KO mice completely lack the function of the targeted gene, while cKO mice allow for tissue-specific or temporal-specific gene deletion. The choice between KO and cKO mice depends on the research question and the specific goals of the study. Wondering what type of KO and cKO mice are out there? Check out Gempharmatech, they have one of the world’s largest libraries of KO & cKO mice for research use.


References

Delgado Caceres M, Pfeifer CG, Docheva D. Understanding Tendons: Lessons from Transgenic Mouse Models. Stem Cells Dev. 2018;27(17):1161-1174. doi:10.1089/scd.2018.0121

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