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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.
Genetically modified mouse models with tissue-specific expression of a reporter gene or Cre recombinase are powerful and commonly-used tools for research. The same transgenic knockin strategies used to make such lines can also be used for tissue-specific expression of almost any gene. For example, lines for controlled expression of a mutant gene sequence can be created using transgenic knockin approaches. Several methods, described below, are available to investigators.
Transgenic knockin techniques for mice went through a period of rapid development in the early 1980s. A common approach starts with the creation of a genetic construct with regulatory and coding sequences, for example a promoter element next to the sequence for a reporter gene. This would be randomly integrated into the mouse genome by injecting linear DNA molecules into fertilized eggs, a method still used today. The injected DNA can be a small artificial expression construct or a long sequence from the human genome, or anything in between. In many cases this method will create a new mouse model with the coding sequence expressed in specific tissues of interest. However multiple integration events must be tested when using this approach since random integration can produce unpredictable results on transgene expression. These varying outcomes result from the random nature of the technique, with the transgene’s expression possibly being affected by the chromosomal region where it integrates.
The strategy of a small promoter sequence driving tissue-specific expression can be improved by using targeted rather than random genomic integration. The Rosa26 locus on chromosome 6 was originally identified as a site where an inserted gene would be ubiquitously expressed during development, and the mouse would suffer no ill effects from the genomic change. The lab of Philippe Soriano identified the locus and demonstrated that it could be specifically targeted to create new transgenic lines without the drawbacks of random transgene integration. Transgenes inserted at the Rosa26 locus are efficiently passed on to the next generation, and each mouse will have an identical level and pattern of expression. Creating a targeted transgenic knockin line initially requires more work and time than simply injecting DNA into fertilized eggs. This is compensated for by the consistent results – only one founder is needed and extensive screening of expression in pups is not necessary.
Another common strategy for obtaining tissue-specific expression in genetically engineered mouse models is knocking in a new sequence so it replaces an endogenous gene. This strategy is preferable for creating reporter lines that most faithfully duplicate the expression pattern of a specific gene. In principle almost any sequence can be used for the knockin, for example a coding sequence incorporating mutations or the coding sequence of a human gene. Often lines made with this method result in disruption of the targeted gene but co-expression of two genes is possible if the right design is used. At ingenious we have the expertise to incorporate these strategies into the design of your next model.
Sooner or later every research program will require the creation of a new tissue-specific expression model. It may be that a required Cre line isn’t already available, or the human and mouse sequences are too different for translational research. The design of a new transgenic knockin model should be guided by careful consideration of the relative advantages of available strategies, particularly the decision between targeted and random transgenesis.
If your lab is considering a new mouse line, contact ingenious to discuss which strategy best fits your requirements.
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