Combating the challenge of off-target Cre effects in research

Cre-lox technology has enabled researchers to manipulate when and where genes are expressed, dramatically increasing their ability to understand how genes function. An even deeper understanding will be possible when this technology is used in combination with the knockout (KO) mice being produced by the KOMP and KOMP2 projects. Most of these mice contain conditionally targetable mutations produced by using Cre-lox technology. Unfortunately, many Cre strains express Cre recombinase in non-target cells and tissues. This unrecognized or unreported activity can confound experimental interpretation and reproducibility. Maximizing the utility of Cre-lox technology will require a sophisticated appreciation of strain-specific Cre-lox limitations and greater access to a diverse and well-characterized set of Cre-driver mouse strains. This article briefly describes Cre-lox technology, The Jackson Laboratory’s high-throughput Cre expression characterization pipeline, and JAX resources that can facilitate research using Cre-lox technology.

Cre-lox technology

Cre-lox technology uses Cre recombinase, an enzyme discovered in a P1 bacteriophage, to translocate, invert or delete a genomic region of interest that is flanked by two unique 34-base pair sequences called loxP sites. Such loxP-flanked genomic regions are commonly referred to as “floxed.” In mouse-based research, Cre-lox technology is used to produce widespread knockouts, conditional knockouts and reporter strains. In almost all cases, a Cre-driver strain (a strain harboring the Cre recombinase transgene) is bred to a loxP strain (a strain with a floxed gene) to delete the floxed gene. Researchers can exploit the technology to control when and where they want to delete a gene.

The JAX Cre characterization pipeline

The experimental reliability of Cre strains very much depends on how well their recombinase activity is characterized. Unfortunately, it is often poorly characterized. In fact, Cre-driver strains frequently exhibit unintended/unreported characteristics: they may express Cre recombinase mosaically, inconsistently, or in non-target cell types and tissues. Cre recombinase may even be toxic to some strains (Heffner et al. 2013). These unintended activities of Cre recombinase can confound research results or cause experiment failure (e.g., if a deleted gene is embryonically lethal). The reported frequency of unintended Cre activity in Cre-driver strains is thought to be only a fraction of the actual frequency.

To provide researchers with well-characterized Cre-driver strains, JAX has designed and implemented a standardized, high-throughput pipeline and is using it to systematically characterize the Cre recombinase activity of the Cre-driver strains in its Cre Repository, which currently consists of nearly 400 strains. The pipeline’s standardized and validated Cre reporter assays ensure that data are efficiently acquired and disseminated to the public.

For each Cre strain, the pipeline identifies Cre-mediated recombination at four time points (E10.5, E15.5, P7, and P56) in a wide range of organs, tissues and cell types. More than 50 of the most widely used Cre strains from JAX’s repository have now been characterized, and several Cre-expression anomalies – including unexpected activity in non-target tissues, inconsistent recombination among littermates, and differential activity in male and female parents-of-origin – have been identified. Researchers should consider these anomalies when designing experiments with these and other Cre strains that could have similar activities (Heffner et al. 2013). The characterization data are publicly available at the Mouse Genome Informatics CrePortal and are regularly updated.