No-Doubt Conditional Knockout™: Certainty Through GFP
Disrupting gene expression has been essential to understanding gene function. As gene targeting technologies have evolved, so has the ability to make these disruptions specific to desired cell types and tissues. Now ingenious provides the next step in gene knockout technology, the No-Doubt Conditional Knockout™ system.
Traditional conditional knockout mouse model systems work by flanking an essential target region of a gene with loxP recombination sites. The respective loxP mouse line is then mated with a Cre recombinase expressing line to induce a recombination event, deleting the region between the loxP sites. Depending on the target cells or tissues, verifying that the recombination event has occurred, and has occurred where desired, can be challenging. Common solutions such as adding/testing conditional reporter alleles/lines for indicating Cre/loxP recombination events requires additional breeding, and does not necessarily provide a reference point at the very same allele to show that the desired genetic manipulation is taking place.
Introducing ingenious’ No-Doubt Conditional Knockout™ System
To address the critical need of researchers to verify the loxP-mediated recombination of their conditional knockout models, and to do so in a reliable manner without increasing timelines, ingenious has developed the No-Doubt Conditional Knockout™ system. Utilizing our proprietary split cassette technology, ingenious is able to include partial sequence segments of GFP in the antisense direction in relation to the loxP sites that flank the target region.
Included in the design is the CAG promoter to drive strong expression of GFP, independent of the target gene’s endogenous promoter and only after the Cre-mediated recombination event has occurred. Prior to the introduction of Cre recombinase, the model is designed to provide normal endogenous gene expression without GFP expression. Furthermore, the segmented GFP has been engineered to prevent functional GFP protein formation in the absence of Cre and prior to Cre-mediated recombination.
After the Cre mediated deletion of the target region, the two partial sequence segments of GFP are brought together for expression from the CAG promoter, thus positively identifying the successful recombination event. This visual cue that the target gene has been disrupted provides an internal, allele-specific control. The neomycin selection cassette has been removed via the use of ingenious’ proprietary FLP ES cells, so no additional electroporations or matings are required for neo removal.
Target region is flanked by loxP recombination sites and partial GFP segments, including a CAG promoter on the antisense strand.
Model is designed to have a wild type expression profile of the target gene with no GFP production prior to the introduction of Cre recombinase.
After the Cre-mediated recombination event, gene expression is disrupted and GFP expression commences under the CAG promoter allowing for positive, no-doubt identification of targeted cells or tissues.
The use of the split GFP cassette under CAG promoter control alleviates concerns about leakiness and weak visual signals.
Positively identify your recombination event via GFP expression.
When using tissue specific Cre recombinase, confirming the recombination event can be challenging. With our No-Doubt Conditional Knockout™ system you can readily confirm the deletion and also determine if there is any leakiness associated or undesired recombination.
GFP is expressed under the CAG promoter on the antisense strand.
Endogenous promoters may be weak or limited in their expression making them unsuitable for reporter production. Placing the GFP under the CAG promoter will offer high expression levels for detection.
Target larger regions with less hassle
Generally, the further the distal loxP site is from the selection cassette the more difficult it becomes to retain during gene targeting. The split-GFP-loxP cassette allows for increased sequence retention and the targeting of larger regions. In our hands, a target distance between loxP sites of virtually any size is possible.
Reduce Breeding Time
Introducing a conditional reporter line to your conditional knockout and Cre recombinase lines can add 2 or more generations of breeding to your project. ingenious’ No-Doubt Conditional Knockout™ system eliminates the need for additional reporter mouse lines.
Combining your No-Doubt Conditional Knockout™ model with Cre expressing mouse lines can come with a number of considerations that need to be addressed. With ingenious’ post project support services we provide the experimental cohorts you need within projected timelines and costs by utilizing our proprietary prediction software.