The Importance of Genotyping Assay Design for Conditional Mouse Lines

1 July 2021

By Chris Smith - Research Assistant at Genetic Research Services


Not all genotyping assays are created equal and, in some cases, using a poorly designed assay can have a significant impact on your mouse lines and your research. This is especially true when it comes to assays for conditional mouse lines.

Conditional lines are used to study the effects of a genetic change (e.g. knocking out a gene) when the genetic change is restricted to particular tissue types, developmental stages, or other situations instead of throughout all the cells of the whole organism. Most conditional mouse lines use the Cre/loxP system to control these genetic changes. This system involves breeding mice that are carrying a ‘Floxed’ allele which has a region of interest in the DNA sequence, such as a gene, flanked by short loxP sequences. The floxed allele often behaves just like the wildtype allele and is usually bred to homozygosity in the line.

Many genotyping assays provided by mouse line repositories for conditional lines are designed to identify only the wildtype and flox alleles to confirm when you have reached homozygous flox mice.

Flox mice are then bred with a line that produces the enzyme ‘Cre’ only in selected tissues or at specific times. In the offspring of this cross the Cre enzyme removes the region of DNA between the loxP sites of the flox allele in specific tissues or at specific times. We refer to this Cre recombined version of the region as the ‘Deleted’ allele.

The trouble is that Cre lines are rarely 100% perfect at restricting the production of Cre to the tissues they’re intended to. The Cre can be ‘leaky’ and be expressed in unintended tissues. If leaky Cre expression occurs in cells from the germline of the animal, then the Deleted allele may form in those cells. If a mouse carrying such a germline deletion is then used for breeding its offspring could inherit the Deleted allele rather than the Flox allele. These offspring then develop into adult mice that have the region of interest permanently deleted in all cells of their body instead of just particular tissues. The mouse line is now a complete knockout for the gene rather than a conditional one which has serious implications for your experimental results.

If your genotyping assay is not designed to identify the Deleted allele or, worse yet, doesn’t distinguish between Flox and Deleted alleles this problem can go undetected. In the worst case scenario this can potential lead to the loss of your conditional mouse line entirely after several generations of breeding.

For these reasons it is important to monitor for the presence of the Deleted allele in your mouse lines to identify germline deletions when they occur and get on top of them before they become a problem. At GRS all of our assays for conditional mouse lines are designed in-house to detect the Deleted allele or any other possible recombination alleles in other conditional systems available. Knowing the complete genotype of your conditional mice helps to protect the genetic integrity of your mouse lines. A more accurate result for the genotype of the mice you are studying can also improve the accuracy and reliability of your experimental observations and data.


If you’d like to find out more about how our genotyping service can assist your research please contact us.


Further Reading

Becher B, Waisman A, Lu LF (2018) Conditional Gene-Targeting in Mice: Problems and Solutions. Immunity. May 15;48(5):835-836.

Liput DJ (2018) Cre-Recombinase Dependent Germline Deletion of a Conditional Allele in the Rgs9cre Mouse Line. Front Neural Circuits. Sep 11;12:68.

Schmidt-Supprian M, Rajewsky K (2007) Vagaries of conditional gene targeting. Nat Immunol. Jul;8(7):665-8.

Song, AJ, & Palmiter, RD (2018) Detecting and Avoiding Problems When Using the Cre-lox System. Trends in genetics : TIG, 34(5), 333–340.

Wu D, Huang Q, Orban PC, Levings MK (2020) Ectopic germline recombination activity of the widely used Foxp3-YFP-Cre mouse: a case report. Immunology. Feb;159(2):231-241.