Understanding how knockout mice are generated is essential for biomedical researchers. Recent advances in genome editing have dramatically streamlined this process, enabling labs to create knockout models more rapidly. Cutting-edge tools now allow faster, more efficient production of gene-disrupted animals, accelerating studies of gene function and disease.

Creating knockout mice involves different strategies depending on research goals. Traditional homologous recombination in embryonic stem (ES) cells and the CRISPR/Cas9 system are the primary methods. Advances in the CRISPR toolbox—including base and prime editors—offer additional options for precise edits. Each method has unique advantages: ES cell targeting allows complex insertions, while CRISPR enables rapid, multiplex editing.

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What is a knockout mouse?

A knockout mouse is a genetically engineered animal with an inactivated gene. Researchers disrupt genes to study effects on development, physiology, or disease. Knockout mice mimic human disorders and reveal gene functions. CRISPR/Cas9 has made creating such models routine, expanding their use in biomedical research.

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Which techniques are commonly used for gene knockout in mice?

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Gene Targeting – A Method for Creating a Knockout Mouse Using ES Cells

Gene targeting via homologous recombination in ES cells remains robust. Researchers design a targeting vector with homology arms flanking the gene, inserting selection markers like neomycin resistance to disrupt the wild-type sequence. The plasmid is electroporated into ES cells, where recombination machinery replaces the native exon with the engineered sequence. Traditional ES cell methods remain effective for complex knockouts.

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Knockout Mice Utilizing ES Cells:

1. Designing and Inserting a Targeting Vector

• Researchers design a targeting vector specific to the gene locus, incorporating genomic fragments flanking target exons and selection markers. The vector is electroporated into ES cells.

2. Identifying Cells that Contain the Knockout Gene

• Cells undergo drug selection and PCR or sequencing-based screening to identify correctly targeted clones. Verified clones are advanced to embryo injection.

3. Generating a Chimeric Mouse

• Verified ES cells are injected into blastocysts, creating chimeric offspring visibly identifiable by mixed coat colors .

4. Breeding Chimeric Mice

• Chimeras are bred with wild-type mice to establish germline transmission. Confirmed via genotyping, offspring carrying the knockout allele are used to create homozygous knockout lines.
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Utilizing CRISPR/Cas9 Technology to Make a Knockout Mouse Model

The CRISPR/Cas9 system uses guide RNA to direct Cas9 endonuclease to specific DNA sites. Delivered as ribonucleoprotein complexes into a zygote, Cas9 introduces double-strand breaks, repaired by non-homologous end joining, resulting in gene-disrupting mutations. Modern protocols use high-fidelity Cas9 variants and chemically modified sgRNAs to minimize off-target effects. Given the high efficiency of the CRISPR/Cas9 system in introducing DNA breaks and in consequence inducing targeted recombination, this makes CRISPR a faster method for certain types of knockout mouse generation.

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Knockout Mice Using CRISPR:

1. Targeting Material Design and Creation

• Researchers select guide RNA sequences using design tools. Synthetic sgRNAs and high-purity Cas9 protein are commercially available, simplifying reagent preparation.

2. Embryo Injection/Electroporation

• CRISPR reagents are delivered via microinjection or electroporation into embryos. Electroporation methods have advanced significantly, enhancing efficiency and throughput.

3. Generation and Screening of Founder Mice

• Injected or electroporated embryos are implanted into surrogate females. Resulting pups (founders) are screened for desired edits, and those with successful germline transmission are bred to establish knockout lines.

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Screen Potential Founders

Unlike gene targeting using ES cells, genome editing efficiency using CRISPR/Cas9 can vary significantly between embryos. To establish a stable mutant line, it is critical to identify founder mice that carry the intended edit in their germline. Although initial genotyping is often performed using DNA from tail or ear biopsies, these tissues may not reflect the genetic composition of the germline due to mosaicism. As a result, a positive result from somatic tissue does not guarantee successful transmission of the mutation. Therefore, generating and screening multiple potential founders is necessary to identify those capable of passing the desired genetic modification to the next generation.

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Breeding F1s

Once viable founder mice are identified, they are mated with wild-type animals to produce F₁ offspring. Because founders can be mosaic—harboring multiple allelic variants—each F₁ line from a given founder should be treated independently. This strategy ensures precise characterization of transmitted mutations. Rigorous validation of each lineage (via sequencing and phenotyping) enables researchers to select F₁ lines that faithfully represent the intended genetic modification.

Validation

Comprehensive validation of the genome-edited model includes sequencing both the mutation site and flanking regions to confirm the intended edit and to rule out potential off-target effects, such as large deletions or rearrangements that may occur during CRISPR editing. Once genetic accuracy is established, functional assays—such as RT-qPCR, Western blotting, or phenotypic assessments—are conducted to verify that the targeted gene has been effectively disrupted and that its expression is absent or significantly diminished.

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How Knockout Mice Differ from Knockin Mice

Although both knockout and knockin mice are genetically engineered models, they serve distinct purposes in functional genomics. Knockout mice are created by deleting or disrupting a gene, rendering it non-functional. This null mutation is used to investigate the physiological roles of specific genes and their involvement in disease processes.

In contrast, knockin mice are designed to introduce precise genetic changes at a specific locus—such as point mutations, reporter tags, or humanized sequences. These models allow researchers to study the effects of defined mutations on protein function, gene regulation, or disease phenotypes. Understanding how knockout models are generated lays the foundation for mastering more complex genome editing techniques, including knockin strategies and conditional alleles.

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