Which Cre Strain Should You Use?

Which Cre Strain Should You Use? Unique in its complexity, modelling the central nervous system (CNS) has long represented a challenge with a complete characterization of constituent cells and circuits arguably unfinished. Indeed, one of the major goals behind the 2013 launch of the BRAIN Initiative was to "identify and provide experimental access to the different brain cell types to determine their roles in health and disease."

Developing Neuroscience Research Models

Multiple mouse models have been developed to further research into the diseases affecting the CNS including models that mimic Alzheimer's disease, Parkinson's disease, ALS, and CNS trauma.

Certain transgenic models have also been developed to employ the Cre/LoxP system. This system allows for tissue-specific inducible knockouts, thereby providing the researcher with control over where and when a gene is modified. These models are used frequently in neuroscience research, and this article will present an introduction to resources that can be used to identify and assess Cre/LoxP transgenic mice prior to making model choices.

Genetically-engineered Cre/loxP mouse models

Cre recombinase (causes recombination) is an integrase originally identified from the bacteriophage P1. As the name suggests, Cre is capable of mediating recombination between loxP (locus of crossover(x) in P1) sites, which are 34 base-pairs in length and asymmetric in nature.

While many new technological advancements have resulted from its discovery1, one of the most complete avenues for cell type-specific access available to researchers today remains the variety of genetically modified models based on Cre/loxP recombination.

Cre-expressing rodent strains are often referred to as "drivers" because they can mediate recombination and thus drive genomic alterations (as well as functional consequences) within a specific cellular population. Such consequences depend highly on the alleles utilized and can include abrogating endogenous expression (e.g, conditional knockout), activating the expression of fluorescent reporter alleles (e.g., lineage tracing), ion channels (e.g., optogenetics), or toxins (e.g., cell-type specific ablation), and allowing viral infection (e.g., transsynaptic tracing).

Cre-driver Resources

Following the first demonstrations that this system worked efficiently in mammalian cells2, an exponential increase had already occurred in the generation and availability of Cre-based resources3 as part of programs such as GENSAT and the NIH Neuroscience Blueprint Cre Driver Network4. The most common strategy used has been to identify and leverage unique patterns of gene expression. This has led to the majority of available Cre drivers, in which expression is under the control of promoter and/or regulatory sequences of individual genes such as transcription factors (e.g., Emx, Cux2, Ascl1, Dlx1) or those related to specific neurotransmitters or neuropeptides (e.g., Chat, Pomc, Th, Dbh).

However, this strategy often marks a broader and more diverse cell population than desired, and "the vast majority of Cre lines express Cre in multiple regions throughout the brain5." For this reason, one should always consult primary literature and independent databases when possible. One comprehensive example of the latter comes from the Transgenic Characterization database, associated with the Allen Brain Atlas and first described in detail by Harris and colleagues, with their analysis of 135 Cre driver lines across 295 separate brain structures6.

Importantly, many such assessments are based on static expression at a particular age or set of ages, and thus temporal changes in gene expression should be strongly considered. When the need for precise temporal control is present, to circumvent undesirable effects or coordinate with experimental manipulations, an inducible Cre driver (e.g., Cre-ER or Cre-ERT2) can and should be considered.

One example that lies in contrast to such cell-type specific Cre drivers, aimed at targeting specific populations, are Cre drivers that function based on neuronal activity through leverage of immediate early genes such as Fos or Arc7. These can be used to provide indiscriminate access to different neurons that are active following a given stimulus.

Further, while an individual Cre driver is often used to impose specificity in conjunction with corresponding "target" or "tool" that would otherwise allow widespread expression (e.g., using the Rosa26 locus), an intersectional approach can also be used to generate increased specificity8.

Selecting a Cre Driver

Many Cre drivers are available from commercial vendors and repositories, such as the Mutant Mouse Resource & Research Centers (MMRRC) and the European Mouse Mutant Archive (EMMA). After a choice is made or during a preliminary assessment, some important technical considerations include:

  • Expression of Cre itself — should always be compared to that of the endogenous gene when possible, as it may not always recapitulate the desired expression pattern.
  • Expression from any targeted or loxP-flanked sequences (e.g., reporter alleles) — should also always be compared to Cre expression, as all loci are not equally recombinogenic. When multiple loxP-flanked alleles are used, their expression (or lack of) should likewise be compared.
  • Generation of appropriate controls — as Cre expression alone may have contributed to cellular or molecular phenotypes, even in the absence of recombination.
  • Maintaining fidelity of targeted (e.g., loxP-flanked) alleles during breeding — as the unintended or leaky expression of various Cre alleles can result in unintended recombination.
Finally, if an appropriate Cre driver cannot be identified or obtained, the potential to generate a unique line using de novo model creation can be considered — and may represent the best solution. Emerging technologies such as Easi-CRISPR have made such prospects more accessible9.

1. Ecker, J. R. et al. The BRAIN Initiative Cell Census Consortium: Lessons Learned toward Generating a Comprehensive Brain Cell Atlas. Neuron 96, 542-557 (2017).
2. Sauer, B. & Henderson, N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc. Natl. Acad. Sci. U.S.A. 85, 5166-5170 (1988).
3. Nagy, A. Cre recombinase: the universal reagent for genome tailoring. genesis 26, 99-109 (2000).
4. Tsien, J. Z. Cre-Lox Neurogenetics: 20 Years of Versatile Applications in Brain Research and Counting.... Front. Genet. 7, 952-7 (2016).
5. He, M. & Huang, Z. J. Genetic approaches to access cell types in mammalian nervous systems. Current Opinion in Neurobiology 50, 109-118 (2018).
6. Harris, J. A. et al. Anatomical characterization of Cre driver mice for neural circuit mapping and manipulation. Front. Neural Circuits 8, 522-17 (2014).
7. DeNardo, L. & Luo, L. ScienceDirect Genetic strategies to access activated neurons. Current Opinion in Neurobiology 45, 121-129 (2017).
8. Huang, Z. J. Toward a Genetic Dissection of Cortical Circuits in the Mouse. Neuron 83, 1284-1302 (2014).
9. Quadros, R. M. et al. Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biol. 18, 92 (2017).