(November 6th, 2015) Have you ever wondered how pure congenic or knockout mice really are? A recent study showed that many unwanted mutations, so-called passenger mutations, accompany the intended genetic modifications, resulting in confounding effects that can have serious impacts on experimental results.
Passenger mutations occur in congenic mouse models, whose modified genetic material comes from a different genetic background to the rest of their genome. Traditionally, knockout and other congenic mice are made by genetically modifying embryonic stem cells (ESCs) in vitro and then injecting these cells into a recipient mouse embryo. The most commonly used ESCs are those from the 129 mouse strain, which are typically transferred into embryos of the B6 strain. The descendants are then backcrossed with B6 mates for 5-10 generations, while selecting for their 129-derived transgene. In this way, DNA from the 129 strain will be slowly eliminated, ideally resulting in one single gene being transferred from the 129 strain onto a uniform B6 background. But the reality is different: pieces of 129 strain DNA, surrounding the transgene, tend to stick around.
Passenger mutations are not at all rare. As a matter of fact, 129 strain congenic mice on a Black 6 background that have been backcrossed for ten generations have more than a 90 percent chance of having around one million base pairs of 129 strain DNA on either side of the transgene. This has huge implications for most experimental results: because when researchers compare their modified mice to control Black 6 mice they may observe phenotypic differences that in fact result from passenger mutations in the 129 strain DNA rather than from changes to their gene of interest.
For example, it has been widely accepted that the gene Casp1 is responsible for triggering an inflammatory and cell death response after being challenged by a foreign organism, a crucial step in septic shock. These findings came out of a study in the mid-1990s, in which Casp1 knockout mice did not go into shock after being challenged with foreign antigens. In 2011, however, a study revealed that protection from this lethal challenge was not because of Casp1, but Casp11, a neighbouring mutated gene from the 129 strain.
This result prompted a group at Ghent University to perform an in-depth exploration of the possible effects of passenger mutations. First, Tom Vanden Berghe and his colleagues studied the sequences of B6 and 129 mouse strains to look for differences that could affect their phenotypes. They found that 1,084 genes in the 129 strain exhibited insertions, deletions, or single-nucleotide polymorphisms, causing their genotype to diverge from B6 mice. Furthermore, the researchers found that, despite intensive backcrossing, nearly all 129-derived genetically modified congenic mice contain multiple passenger mutations.
The Ghent group then went a step further and brought their in silico findings to the lab. They specifically looked at mice with the Casp11-inactivating mutation and found that 86 mouse models had them in genomic regions close to the targeted gene. Thus, all of these mice could be resistant to lethal shock for an “unexpected” reason. Indeed, when they examined mice null for the Mpp13 gene, they found that these mice were resistant to shock, probably due to the Casp11 passenger mutation. Interestingly, another laboratory that had backcrossed the B6 transgenic mice for more generations found that the same Mpp13-null mice had lost their protection against shock. This example clearly illustrates how inconsistent phenotypes can be between different labs that are supposedly using the same congenic mouse line.
From their bioinformatics analysis, Vanden Berghe created ‘Me-PaMuFind’, a web-based tool that allows researchers to instantly and conveniently identify all 129-specific passenger mutations for several congenic mice. With this web-based tool, the Ghent group hope that new studies will be carried out more carefully, but also that some of the old dogmas will be revisited.
This eye-opening study clearly raises the alarm about the insidious nature of passenger mutations, but most of all, the dangers of ignoring their existence!