4 essential steps to verify your Cre-lox model

Bipolar neurons

You’ve created mice that carry a Cre-conditional (“floxed”) allele in your target gene.  You’ve bred them with mice that should express Cre in your target tissue. Here we go over the four essential steps you need to take to validate your tissue-specific KO model, and the reasons why you may still detect protein expression.

Step 1: Genotype the mice!

Whenever you observe unexpected phenotypes when working with mice, your first action should be to verify the genotypes of your animals. If you prepared protein extracts from a specific tissue, keep a frozen sample in your freezer so that you can harvest genomic DNA from it and re-verify the genotypes of the mice.

With tissue-specific Cre-lox KO models, the genotype of the mice that you generally want to evaluate is homozygous for your floxed allele (fl/fl) and positive for Cre (cre+). (Typically, only one copy of the cre transgene is required for efficient recombination.) As we have discussed in a previous blog post, two rounds of breeding are necessary to produce such mice, even if the floxed and Cre parentals are homozygous for their respective alleles. Therefore, if the mice from which you harvest the tissue are the offspring of your first Cre-lox cross, then they are heterozygous for your floxed allele and still express functional (wild-type) protein.

Step 2: Genotype genomic DNA from the target tissue

If you only genotyped the mice via a tail tip or ear punch biopsy, it is also important to check their genotype in the target tissue using fresh or frozen tissue sample mentioned above. This requires that you have a PCR assay that will distinguish the recombined, KO allele from the unrecombined, floxed allele. Please note that most JAX genotyping protocols for floxed mice have not been designed for this purpose; therefore, you may need to check the literature for such an assay or design your own. Alternatively, you can genotype the mice by Southern blot with an appropriate DNA probe.

Keep in mind that many tissue preparations contain heterogeneous mixtures of cell types. Therefore, you may still observe some level of unrecombined product even if Cre has recombined the floxed allele in your target cell type.

Step 3: Check for cre expression in the target tissue

If your genotyping analysis indicates that the floxed alleles are intact, and you are not detecting the recombined alleles, you need to seriously evaluate the tissue for cre expression.There are several ways to do this:

  • Check for cre mRNA transcripts using quantitative RT-PCR or Northern analysis of mRNA harvested from the target tissue. This probably is the easiest and most straightforward approach.
  • Evaluate Cre protein via immunohistochemistry (IHC) or by immunoblot. Be sure to choose a Cre antibody with demonstrated performance in these techniques. If you are using IHC, be sure that you use appropriate fixation conditions for cytosolic proteins.
  • Breed your cre mice to a Cre reporter strain, for example strains with either a beta-galactosidase (lacZ) or a fluorescent reporter, and check for Cre recombination in the cre+ offspring.

How do lacZ reporter strains work?  Many lacZ reporter strains have a loxP-flanked DNA stop codon that prevents the expression of lacZ. The reporter gene will only be expressed when and where Cre is functional. In this example the pattern of Cre recombinase activity  was evaluated early in development (E15.5) of  the offspring of a female reporter strain B6.129S4-Gt(ROSA)26Sor<tm1Sor>/J (003474) bred to a male strain B6.Cg-Tg(Nes-cre)1Kln/J (003771).

These approaches can also be used to rule out the fact that cre expression may be down-regulated or even silenced over time.  Unfortunately this phenomenon may happen when breeding cre mice, particularly cre transgenics created by classic microinjection.

Step 4: Evaluate mRNA expression from the target gene directly

Perhaps the best way to validate whether your flox/flox, cre+ mice are still making a target transcript is to evaluate mRNA expression from the targeted gene directly.    Depending on your target gene and how you designed your floxed allele, the recombined allele may still produce transcripts that translate into, for example, a truncated protein product.   Be sure 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.

Any particular cre-expressing mice will not necessarily recombine all floxed alleles with equal efficiency (see, for example, Vooijs M et al. 2001). Therefore, if your floxed allele is untested, it may just be one that is resistant to recombination.  Explanations for such phenomenon could be the following:

the position of your target gene in the genome
the position of the loxP sites in your construct
the target gene expression level in the target tissue
a point mutation on either loxP site

If this is the case, you may need to go back to the drawing board to redesign your floxed alleles.