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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 mice were first made with a technique that’s still useful today: by randomly integrating foreign DNA into the mouse genome. This method can be used with a minimum amount of specialized training and equipment. A plasmid containing DNA to be knocked in can be created using the same process most labs use every day. Injecting DNA into mouse embryos requires practice and is a standard skill for technicians in core facilities. However, the ease of creating such mouse models using random DNA insertion is balanced by unpredictable results. It was quickly realized that a more consistent strategy was needed and the discovery of the Rosa26 locus enabled a new and more consistent method for generating knock-in mice.
Rosa26 refers to a specific site on the mouse chromosome 6. It was identified in a screen that was designed to find places in the genome where the insertion of foreign DNA would not have unpredictable results. Unpredictable results in this case could mean two things – either unwanted effects on the health of the mouse or unpredictable expression of the foreign DNA. Carefully targeting the foreign DNA into the Rosa26 locus seems to have essentially no effect on the mouse’s health. In addition, the DNA that’s inserted will have a consistent expression pattern. With the older method of randomly inserting DNA into the genome, it’s possible for the new DNA to have an unpredictable expression pattern. This can happen if it’s inserted near another gene, and that gene’s promoter influences the inserted DNA. With the Rosa26 locus this is not the case.
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Rosa26 is used for knockin mice when consistent results are the most important criteria. A Rosa26 knockin mouse will have a single copy of the foreign DNA inserted into a known site in the genome. The DNA will be expressed in a consistent way and that won’t change from generation to generation as the mice are bred. In contrast, with the random insertion method multiple copies of the DNA will be inserted. Many more mice will need to be bred and tested until the new line has a stable expression pattern, and that pattern may not be exactly what was intended. Targeting to the Rosa26 locus is the clear choice for consistent results.
The long track record of success using the Rosa26 locus, and the consistent results that it can obtain, are balanced by a more complex process for creating models. A targeted transgenic knockin requires additional steps to create as well as more technical knowledge. The standard method is to create a genetic targeting construct which will be larger than the plasmids most labs are used to working with. Embryonic stem cells must be cultured, modified, and screened. Correctly modified cells are then injected into mouse embryos to make chimeras. Partnering with an experienced team streamlines all these steps and can prove invaluable when taking advantage of the consistent, reliable results that Rosa26 knockins can provide.
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