The use of Cre recombinase and loxP sites in genetically modified mouse models allows for tissue specific control over genetic modifications, enhancing our understanding of gene functions. Hundreds of different Cre mice are already available, with different patterns of Cre expression allowing conditional knockout in different tissues. If the Cre mice you need aren’t already available a new line can be created by two techniques: random transgene integration and targeted homologous recombination. Both of these approaches, however, have limitations.
Cre recombinase can be toxic in ES cells, causing difficulties when linking expression to genes that are active during embryonic time points. Random integration transgenic mice produced via pronuclear injection have been used as an alternative to avoid embryonic stem cell lethality; however, these models do not faithfully reflect endogenous gene expression patterns. To solve this problem, an approach is needed that enables native gene expression patterns while avoiding ES cell toxicity.
ingenious’ Split Cre Technology
By splitting Cre recombinase in half with an FRT-flanked neomycin selection cassette, we developed a gene-targeted ES cell approach for generating your mouse line without the worry of ES cell toxicity.
The neomycin cassette prevents expression of Cre recombinase until the introduction of FLP recombinase, allowing for the generation of properly targeted ES cells without the risk of toxicity. Once chimera mice are produced, mating them to a constitutively active FLP recombinase mouse line results in removal of the neomycin selection cassette and the safe expression of Cre. Our split design can be utilized for gene replacement or co-expression knockin, to generate Cre recombinase expressing mouse lines that follow the native expression patterns of any target gene regardless of expression location or timepoints.
In addition to specific, gene-targeted Cre knockins, we can add a reporter to your targeting design for visualizing expression.
Split Cre System Overview
Cre recombinase expression is blocked during the embryonic stem cell stage where it may be toxic.
Removal of the FRT-flanked neomycin selection cassette allows for safe expression of the recombinase regardless of expression location or timepoints.
Split Cre System Benefits
Cre expressing mouse lines that were previously unavailable due to toxicity can now be generated without risk.
After You Receive Your Mice
Once you receive your Split Cre mouse model from ingenious, your next step will be to generate a cohort of mice of a certain age and sex, which you can then utilize for your experiments. The generation of experimental cohorts can be a challenging process requiring multiple matings, strategic genotyping, and calculated colony scale up. ingenious offers prediction tools for developing the cohorts you need, as well as cryopreservation services to protect your investment. Read more about our post project support services.