How to Verify Your Cre-lox Model

Now that the genetically engineered mice that carry a Cre-conditional (“floxed”) allele in target gene are at your hand. And the mice are supposed to express Cre in target tissue. The validation of your Cre-lox model become very indispensable, so here we advise you of some essential steps.

Genotype the mice

When unexpected phenotypes are observed during your working with mice, the verification of the genotypes should be performed immediately! Don’t forget to keep some frozen sample in order to harvest genomic DNA from it and re-verify the genotypes of the mice when protein extract of specific tissue are prepared.

In general, for tissue-specific Cre-lox KO models, the genotype of the mice you desire is homozygous for the floxed allele (fl/fl) and Cre-expressing (Cre+). In theory, mere one copy of Cre is sufficient to guarantee the recombination. To achieve such mice, two rounds of breeding are required, even if the floxed and Cre parentals are homozygous for their respective alleles.

Genotype the target tissue

If the genotyping of the mice is merely performed through a tail tip or ear punch biopsy, the verification of their genotype in the target tissue using fresh or above-mentioned frozen tissue sample is indispensable. Generally speaking, a PCR assay is commonly used to distinguish the recombined, KO allele from the unrecombined, floxed allele. Please check http://www.modelorg.us/faq.html?page=1 for more information about the verification protocol using PCR. Besides, Southern blot using probes will also be applicable to the genotyping.

Keep in mind that, since some heterogeneous mixtures of cell types exist in many tissue preparations, some unrecombined products will be observed even if Cre has recombined the floxed allele in your target cell type.

Evaluate Cre expression in the target tissue

In some cases, even If the genotyping indicates that the floxed alleles are intact, the recombined alleles are undetected. Cre expression need to be evaluated in the target tissue. There are several ways to do this:

1.Evaluate Cre mRNA transcripts via RT-PCR or Northern blot using mRNA derived from the target tissue.

2.Evaluate Cre protein using IHC or Western blot. A Cre antibody applicable to both IHC and WB is recommended. Keep in mind that appropriate fixation conditions is required for cytosolic proteins when using IHC.

3.Evaluate Cre expression based on reporter gene expression through crossbreeding Cre mice and a Cre reporter strain, such as, strains with either a beta-galactosidase (lacZ) or a fluorescent reporter, and analyse Cre recombination in the Cre+ offspring.

So how do Cre reporter strains work in the evaluation of Cre expression?  

Cre reporter strains are usually characterized by loxP-flanked stop codon that stems the expression of reporter gene. The reporter gene will only be expressed on condition that functional Cre exists.

Besides, above-mentioned methods can also be applied to determining if Cre expression may be down-regulated or silenced over a long period of time. In some cases, it may happen to Cre mice, especially transgenic Cre mice generated using conventional microinjection.

Evaluate mRNA level of the target gene

Evaluation of mRNA level of the target gene could be the most reliable way to certify whether your flox/flox, Cre+ mice are still making a target transcript. Depending on the target gene and how you designed the floxed allele, the recombined allele may still produce transcripts that translate into, for example, a truncated protein product. Keep in mind that to use qPCR primers or probes from regions upstream, downstream and from within the deleted exons to determine whether any transcript that you might detect is full-length, alternatively spliced or truncated.

In general, Cre-expressing mice could recombine all floxed alleles without equal efficiency. So, in some cases, you may encounter untested floxed allele, and it might be caused by the resistance of floxed allele to recombination. Such phenomenon could be accounted for as follows:

• the position of the target gene

• the position of the loxP sites

• the target gene expression level in the target tissue

• a point mutation on either loxP site

Once the cause is confirmed, you are strongly advised to redesign the floxed alleles.