It’s far too common in the lab to be given a set of primers and a protocol and be asked to genotype without any clear specifics on the animal strain itself. While you may end up with bands at the right sizes, that doesn’t always guarantee that you have the exact mutation you think you do. Here are three things that we believe will be helpful to keep in mind when starting to genotype any line.
1. Not all mutations are the same
It’s essential to know the details of each mutation you work with in the lab. Not all TLR4 knockouts are the same, and not even all SNP strains are the same. Knowing the genetics of the mutation itself helps promote reproducibility across your experiments and provides a starting point for troubleshooting when things don’t go as expected. It may seem trivial but knowing whether that neomycin cassette disrupts exon 2 or exon 3, can have a large impact on both your genotyping and experimental results. Before choosing a strain, consider reading peer-reviewed publications, speaking with colleagues, and/or referring to vendor websites to ensure that you’re aware of known phenotypes, health status, and breeding considerations.
2. Mix-ups happen to the best of us
When the only thing distinguishing one animal from another is an ear punch, it’s easy to confuse animals when setting up breeders. On top of that, genotypes scribbled in a lab notebook during incubation periods can be recorded incorrectly. As if the lab needs any other variables, while it’s rare, animal vendors do also make mistakes and ship the wrong genotype or strain to institutions. As a result, it’s incredibly important to verify the genotypes of animals when performing an experiment, or at absolute minimum every few generations. This prevents time and resources from being wasted breeding the wrong animals and can prevent unexpected experimental outcomes. Regular genotyping can be an easy way to check your work, even if you think you’re just trying to maintain a colony of a known background. This is particularly essential if the lab uses multiple strains that target the same gene. With up-to-date records that are easily accessible, you can prevent mistaking one line for another and wasting months of valuable time.
3. Leaky Cre is more common than you think
Conditional models have become a powerful method to fine-tune and heavily control genetic testing. However, just because a Cre line is only expected to recombine in one cell type, does not mean that the Cre does not also function in off-target tissues. While it’s a fact that may hide in the back of your mind, it’s much more common to have a leaky Cre than you may think. It’s important to properly characterize any new Cre line to determine any off-target tissues with Cre expression that may confound experimental results. Leaky Cre can also result in undesired mosaic animals, with recombination in some cells of a tissue and no recombination in other cells. For more information on leaky Cre, check out this article from Jax.