Name: knockout mice and gene replacement
Uploaded: 2026-08-26T08:20:04.844Z
Duration: 2 min 29 s
Channel: JoVE

Scientists can inactivate, or &#x201C;knock out,&#x201D; a gene of interest in mice to study its function. This is usually done by replacing the gene with a targeting vector, which is an engineered DNA sequence.The targeting vector contains sequences homologous to the regions before and after the gene. These matching regions are called homology arms.The targeting vector also contains marker genes that help identify cells in which correct gene replacement has happened.These include a positive selection marker, such as the neomycin resistance gene, NeoR, located in the middle of the targeting vector. This marker provides resistance to the antibiotic neomycin.A negative selection marker, such as the thymidine kinase gene, or TK, is located outside the homologous region. This marker makes cells sensitive to the drug ganciclovir.When introduced into mouse embryonic stem cells, the vector replaces the target gene through homologous recombination between its homology arms and the matching DNA sequences in the genome.After homologous recombination, the target gene is replaced by the NeoR gene, while the TK gene is not incorporated because it lies outside the homologous region.As a result, cells that undergo correct homologous recombination contain NeoR but not TK. These cells survive treatment with both neomycin and ganciclovir, allowing researchers to select them in culture.These cells are then injected into early-stage mouse embryos, which are implanted into the uterus of a female mouse. The resulting mice are chimeric because their tissues contain cells derived from both the original embryo and the genetically modified embryonic stem cells.When the chimeric mouse is bred, some offspring inherit one copy of the knockout allele and are called heterozygous knockout mice.Breeding these mice can produce homozygous knockout mice, in which both copies of the target gene are inactivated.

in-vitro mutagenesis (gene knockouts)

In-vitro Mutagenesis (Gene Knockouts)

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or &#x201C;knocked out,&#x201D; by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson&#x2019;s disease, and diabetes.

<h4>The Process</h4>

Genes can be randomly knocked out, or specific genes can be targeted. To knock out a particular gene, an engineered piece of DNA called a targeting vector is used to replace the normal gene, thereby inactivating it.

Targeting vectors have sequences on each end that are identical&#x2014;or homologous&#x2014; to the sequences flanking each side of the gene of interest. These homologous sequences allow the targeting vector to replace the gene through homologous recombination&#x2014;a process that occurs naturally between DNA with similar sequences during meiosis.

The targeting vector is introduced into mouse embryonic stem cells in culture, using methods such as electroporation&#x2014;use of electric pulses to temporarily create pores in the cell membrane. Typically, to identify cells where the vector has properly replaced the gene, it is designed to include a positive selection marker&#x2014;such as the gene for neomycin resistance (NeoR)&#x2014;between the homologous regions; and a negative selection marker&#x2014;such as the gene for viral thymidine kinase (TK)&#x2014;after one of the homologous regions.

The cells are exposed to neomycin, and only those that have incorporated the vector into their DNA will survive because they have the NeoR gene. Also, cells, where the vector has replaced the targeted gene through homologous recombination, will not have the TK gene, allowing them to survive in the presence of the drug ganciclovir. Therefore, exposure to ganciclovir is used to eliminate cells that have the vector randomly inserted into their genome, because these cells will have the TK gene.

The cells with the gene properly knocked out are then inserted into a mouse embryo, which is implanted into the uterus of a female, where it develops until birth. The resulting mouse is a chimera&#x2014;meaning it is composed of a mixture of cells&#x2014;some with normal DNA from the embryo, and some with the gene knocked out on one chromosome from the engineered cells. These mice are bred, and offspring containing the gene in their germline are further crossbred to create a line of mice where every cell is homozygous for the knockout. These knockout mice can then be used to study gene function.

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or &#x201C;knocked out,&#x201D; by creating ...

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KEY TERMS AND CONCEPTS

knockout mice and gene replacement

Knockout mice help researchers study gene function by showing what happens when a gene is turned off. These engineered animals are also useful for modeling human diseases such as cancer, Parkinson’s disease, and diabetes.

A specific gene can be targeted by using a targeting vector, which is a piece of engineered DNA that replaces the normal gene and inactivates it. The vector has matching DNA sequences on both ends. These homologous sequences let the vector swap in for the target gene through homologous recombination, a natural process that happens between similar DNA sequences during meiosis.

Scientists introduce the targeting vector into mouse embryonic stem cells in culture, often by electroporation. Electroporation uses electric pulses to make temporary pores in the cell membrane. The vector is usually built with a positive selection marker, such as the neomycin resistance gene (NeoR), between the homologous regions. It also includes a negative selection marker, such as viral thymidine kinase (TK), after one homologous region.

The cells are treated with neomycin, and only cells that took up the vector survive because they carry NeoR. Cells in which the vector replaced the target gene by homologous recombination survive ganciclovir treatment because they do not have TK. Ganciclovir removes cells in which the vector inserted randomly into the genome, since those cells still carry TK.

The correctly targeted cells are then placed into a mouse embryo. The embryo is implanted into the uterus of a female and allowed to develop until birth. The mouse that results is a chimera, which means it contains a mixture of cells. Some cells come from the original embryo, and some come from the engineered stem cells with one knocked-out chromosome.

These mice are bred, and offspring with the knockout in their germline are crossbred again. This creates a mouse line in which every cell is homozygous for the knockout. Researchers can then use these knockout mice to study the function of the gene.

Knockout Mice and Gene Replacement

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Knockout mice help researchers study gene function by showing what happens when a gene is turned off. These engineered animals are also useful for modeling human diseases such as cancer, Parkinson’s disease, and diabetes. A specific gene can be targeted by using a targeting vector, which is a piece of engineered DNA that replaces the normal gene and inactivates it. The vector has matching DNA sequences on both ends. These homologous sequences let the vector swap in for the target gene through...

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key terms and concepts

See how targeting vectors replace a gene in mouse stem cells, how selection markers work, and how knockout mice are bred for gene function studies.

Knockout Mice and Gene Replacement | JoVE K12

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Knockout Mice and Gene Replacement

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