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Knockout mouse
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What is a knockout mouse?
A knockout mouse is a genetically engineered laboratory mouse with a specific gene inactivated by introducing foreign DNA. Studying knockout mice helps researchers understand gene functions and their roles in diseases because mice share many genes with humans.
Generating knockout mice involves techniques like homologous recombination and gene trapping, using embryonic stem cells to replace or disrupt target genes with foreign DNA. This DNA often includes a reporter gene for tracking.
These models help in understanding diseases like arthritis, cancer, and cystic fibrosis, and in developing new drug therapies. However, this method has limitations, such as the fact that the inactivation of certain genes can be lethal.
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How are knockout mice different from regular mice?
A knockout mouse is a genetically engineered lab mouse with a specific gene inactivated through the introduction of artificial DNA. This allows scientists to study the function of individual genes by observing changes in the mouse's traits.
Regular mice have their complete set of genes, while knockout mice have a specific gene intentionally disabled. Researchers use knockout mice to model human diseases and to study the effects of treatments and genetic changes. Because mice and humans share many genes, studying knockout mice can provide insights into the roles of specific genes in human diseases. These engineered mice help advance the understanding of gene function and hereditary diseases.
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How do scientists create a knockout mouse?
A knockout mouse is a genetically engineered mouse with a specific gene inactivated by a foreign DNA sequence. Scientists use homologous recombination or gene trapping to achieve this. Both methods involve using mouse embryonic stem cells. In homologous recombination, artificial DNA replaces the target gene, inactivating it. Gene trapping randomly incorporates foreign DNA into any gene, disrupting its function.
To create a knockout mouse, scientists introduce modified embryonic stem cells into early mouse embryos, which are then implanted into a surrogate female. The resulting pups are bred over generations to produce true knockout mice. These mice help scientists understand gene functions and their roles in human diseases.
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Why do researchers use knockout mice in experiments?
Researchers use knockout mice in experiments because they share genetic similarities with humans, allowing scientists to model and study human diseases. These mice are genetically engineered to have specific genes inactivated, which helps researchers understand the roles of these genes in various diseases.
With short life cycles, mice enable scientists to observe the effects of genetic changes or treatments across an entire lifespan or multiple generations quickly. Studying knockout mice helps in understanding gene function, hereditary diseases, and potential gene therapies. They also serve as a platform for developing and testing new drug therapies.
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What kinds of diseases or conditions do knockout mice help study?
Knockout mice are genetically engineered to help scientists study the function of genes and their roles in diseases. Because mice share many genes with humans, these models can provide insights into human health.
Knockout mice have been developed to study various human diseases and disorders, including arthritis, atherosclerosis, cancer, cystic fibrosis, diabetes mellitus, hypertension, Parkinson's disease, and thalassemia. They have also advanced the understanding of genes involved in fetal development and congenital disorders and serve as a platform for developing and testing new drug therapies.
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What are some limitations or challenges of using knockout mice in research?
Knockout mice, laboratory mice genetically engineered to have a specific gene inactivated, offer insights into gene function and human diseases because mice and humans share many genes. However, using knockout mice in research has limitations. Inactivation of about 15% of genes via targeted knockout is lethal, preventing altered embryos from developing into adult mice. In these cases, the functions of those genes cannot be fully studied.
Additionally, knocking out a gene may not produce any phenotypic change, and changes observed in mouse models may differ from those observed in humans when the same gene is inactivated in both species. Despite these drawbacks, knockout mice remain a highly effective means for studying gene function in living animals.
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Knockout mouse, genetically engineered laboratory mouse (Mus musculus) in which a specific gene has been inactivated, or “knocked out,” by the introduction of a foreign (artificial) DNA sequence. Knockout mice exhibit modifications in phenotype (observable traits) and thereby provide important clues about the function of individual genes. Mice and humans share many genes in common. Hence, information from knockout mouse models can shed light on the biological roles of specific genes as well as on the involvement of those genes in human disease.
Generation of knockout mice
Technologies used to generate knockout mice include homologous recombination (or gene targeting) and gene trapping. Both approaches involve the use of mouse embryonic stem (ES) cells that are isolated from mouse embryos at about four days following fertilization. In the first approach, homologous recombination, artificial DNA with flanking sequences that are homologous (identical) to those occurring upstream and downstream of the target gene DNA sequence is introduced into the nucleus of a mouse ES cell. The ES cell recognizes the homologous flanking sequences and exchanges the existing target gene DNA for segments of the foreign DNA. The foreign DNA, however, is inactive. Thus, its incorporation into the ES cell genome inactivates, or knocks out, the target gene. The foreign DNA typically is engineered to carry a reporter gene, which marks or tags the location of the existing gene, enabling researchers to track its presence in the mouse cell genome as the cells replicate. Homologous recombination is the method of choice when it is required that a specific allele (gene) be replaced by an engineered DNA sequence without affecting any other genes in the genome.
Gene trapping also makes use of artificial DNA that carries a reporter gene. However, rather than targeting a specific gene, the foreign DNA is randomly incorporated into any gene in the mouse ES cell genome. The foreign sequence prevents the disrupted gene from encoding its protein products, thereby rendering the gene inactive. Activation of the reporter gene upon incorporation into the ES cell genome enables researchers to track gene activity and deduce the disrupted gene’s function.
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Artificial DNA sequences typically are introduced into mouse ES cells using a retrovirus or other viral vector, and the modified ES cells are then grown in cell cultures. After a few days, the cells are injected into early mouse embryos, which subsequently are implanted into the uterus of a surrogate female, where they eventually develop and are carried to term. The mouse pups born in this manner contain both modified and nonmodified tissue and thus are not complete knockout mice. In order to generate a pair of true knockout mice (homologous knockouts), the mice must be bred over several generations. Initially, the animals are crossbred with other mice to produce heterozygote individuals (mice with one copy of the target gene); heterozygote animals are then interbred to produce homozygotes.
Applications
Homologous recombination and gene trapping allow researchers to study mammalian physiology in great depth. Knockout mouse models also give a better understanding of the role of similar genes involved in human diseases. Knockout mouse models have been developed for multiple human diseases and disorders, including arthritis, atherosclerosis, cancer, cystic fibrosis, diabetes mellitus, hypertension, Parkinson disease, and thalassemia. The specific targeting of genes with homologous recombination has shed light on the role of numerous genes involved in fetal development and has helped advanced understanding of the causes of different human congenital disorders. The involvement of genes in mammalian organ development and establishment of body plan has been studied thoroughly, owing largely to the development of knockout mice. Knockout animal models also have provided a platform on which to develop and test novel drug therapies.
There are limitations, however, to homologous recombination and gene trapping. For about 15 percent of genes, inactivation via targeted knockout is lethal, preventing altered embryos from developing into adult mice. Thus, the functions of those genes cannot be studied to the full extent, and their roles in human biology and disease cannot be established through knockout approaches in animals. In some cases, the genes play different roles in embryological stages and adulthood. Furthermore, knocking out a gene may not produce any phenotypic change, and the changes observed in mouse models may be quite different from those observed in humans when the same gene is inactivated in both species.
Despite the drawbacks, homologous recombination and gene trapping have proven to be highly effective means for studying gene function in living animals. The successful production of a wide array of knockout mouse models has resulted in the generation of a vast pool of knowledge that has aided advances in the treatment and prevention of human disease.
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