Targeted transgenesis allows for the integration of exogenous DNA sequences into specific – or targeted – sites within the mouse genome. This is advantageous as it avoids many of the risks associated with random integration transgenesis, including insertional mutagenesis and the potential for multiple integrations.
Taconic can generate targeted transgenic mice using several different methodologies and can utilize many different and well-characterized safe harbor sites. The most commonly used safe harbor site is ROSA26, which was originally identified in a promoter trap screen and is located on mouse chromosome 6. The most common method used by Taconic to insert targeted transgenes into the ROSA26 locus is recombinase-mediated cassette exchange (RMCE). This is possible as Taconic maintains a custom line of embryonic stem cells (ESCs) on a C57BL/6NTac genetic background, in which a landing pad (or appropriate recombinase sites, along with other genetic elements) was previously inserted. By transfecting these custom ESCs with an appropriate targeting vector, as well as providing the appropriate recombinase, it is possible to achieve targeted transgenesis at high frequency. This, in turn, helps reduce the burden of screening and genetic validation that must be performed for this project type.
Targeted transgenesis is commonly used to drive the constitutive expression of a particular gene product, by placing the appropriate coding sequence under control of a ubiquitous promoter. Many different promoters have been utilized for this purpose, though a common choice is the strong CAG promoter.
Conditional (e.g., tissue-specific) or inducible expression may also be achieved using targeted transgenesis, though the choice of safe harbor should be made carefully for such projects. Tissue-specific promoters may or may not function as intended in safe harbor sites, so it is common to allow for conditional expression from a safe harbor site by instead using a "lox-stop-lox" element, which places two loxP sites on either side of sequences which will stop transcription. When this element is inserted between a strong ubiquitous promoter and a coding sequence of interest, expression will only be achieved when the lox-stop-lox element is removed. And this may be accomplished by using a Cre driver in which tissue-specific expression of Cre recombinase comes from another locus. Inducible changes in expression are similarly often pursued by placing a transgene in a safe harbor locus while under the control of a promoter whose activity may be controlled using the tetracyline system (e.g., administration of doxycycline to control transactivators encoded at a separate location in the genome).
