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"I’ve been working with iTL over the past 5 years in the production of 3 different genetically altered mice. Not only did iTL help in the design of the mice, […]” - Raghu Mirmira, MD, PhD University of Chicago.
Knock-in and knockout mice are both kinds of genetically modified mouse models used by researchers to study areas such as human disease, genetics, and basic biology. These mouse models are used in conjunction with human cells that can be cultured in the lab and also genetically modified. Growing mutated human cells in a dish can be highly informative for figuring out the effects of the mutation. However in many cases the full effects of a mutation can’t be seen unless the cells are living inside an animal or person. This is where animal models such as knockin mice come into the picture. The biology of the mouse is very similar to that of humans in most respects so a mutation in the mouse’s cells will usually have the same effect as it would in a person’s. Scientists who want to study a mutation will evaluate different strategies for making a genetically modified mouse model, for example looking at knockin vs knockout modifications. Creating the best model is a crucial early step in a successful research project.
The processes for making knockin mice and knockout mice are similar in many ways and require special skills, tools, and reagents. A major difference in knockin vs knockout is that a knockout mutation is always targeted to a precise spot in the mouse’s genome. This is because the goal of a knockout mouse model is to prevent a gene of interest from functioning so the genetic modification must happen in that gene’s sequence. A knockin mutation can be targeted but it’s also possible to insert a knockin sequence randomly into the genome and find out later where it ended up. This method was used to generate some of the very first genetically modified mouse models in the early 1980s, which had new genetic sequences randomly inserted into their genomes.
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It’s relatively easy to describe a knockout mouse model because there are only a few methods to make them and the goal is always the same: inactivating a target gene. The most common method today uses CRISPR technology to make a small change near the start of the gene’s coding sequence. Either a small part of the gene is deleted or a small new sequence is added. If the correct modification is made this has the effect of interrupting the gene expression process. The cell’s machinery makes an RNA copy of the mutated DNA, then recognizes something is wrong and destroys the RNA without translating it into protein. Older ways of making knockout mice are broadly similar. A specific part of the gene is removed, sometimes in combination with the addition of a new blocking sequence. With any of these methods the modified copy of the gene is permanently inactivated.
The picture is more complex when talking about knockin vs knockout. Knockin mice may have a new genetic sequence added that is turned on in all cells, or only certain cells, or in response to mice receiving a specific drug. The knockin gene might cause a disease in the mice, or correct a problem caused by a different mutation, or mark certain cells with a fluorescent protein label. As mentioned previously the knockin sequence can be put into a specific location or added to a random genomic site. A particularly exciting kind of knockin replaces part of the mouse’s genome with a human DNA sequence. This enables the study of the human gene in the context of a living mouse and knockin mice with human genes will be a powerful tool for researchers. The knockin approach is incredibly powerful because it’s so versatile and a new knockout or knockin model can be a valuable addition to a new or ongoing research project.
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