Two lines of transgenic mice expressing cre-recombinase exhibit increased seizure susceptibility

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Summary

Conditional mouse models based on the Cre-recombinase (Cre)-loxP method are a powerful tool for determining the spatial and temporal function of genes in neuroscience research. The Emx1-Cre conditional model is designed to drive Cre expression in a predominantly excitatory neuron specific manner and the Dlx5/6-Cre mouse expresses Cre predominantly in cortical inhibitory neurons. The mouse models expressing the Cre transgene are healthy, active and have no overt behavioural or brain histological phenotypes. Subcutaneous pentylenetetrazol (scPTZ) is a proconvulsant frequently used to probe neuronal network excitability. In both the Emx1-Cre and Dlx5/6-Cre conditional mouse models the latency to scPTZ-induced seizures was significantly shorter than for their wild-type littermates. This shows that mouse models carrying the Cre transgene alone can have significant behavioural phenotypes. This may act as a confound to the interpretation of data obtained from crosses with loxP-flanked targets especially in the context of epilepsy phenotypes. These data highlight that appropriate control experiments that compare wild-type mice to those that carry the cre-transgene but not the loxP-flanked target are essential when using this method.

Introduction

Conditional genetic targeting in mice allows for the spatio-temporal control of the expression or deletion of genes-of-interest. The primary method used to achieve this is based on Cre-recombinase (Cre), an enzyme isolated from bacteriophage P1 (Glaser et al., 2005). Cre specifically catalyzes recombination between two loxP recognition sites resulting in the irreversible excision of the encompassed DNA segment. There are no loxP recognition sites within mammalian genomes, which accounts for the specificity of this gene-targeting strategy in genetically modified mice. The Cre–loxP method has become a standard experimental method used widely within neuroscience research (for review see Gaveriaux-Ruff and Kieffer, 2007, Morozov, 2008). However, the technique is not without limitations. Cre toxicity is well documented (Schmidt-Supprian and Rajewsky, 2007). Permanent high levels of Cre can arrest growth and induce chromosomal abnormalities in fibroblasts (Loonstra et al., 2001). Similarly, Cre caused significant damage to genomic DNA in transgenic mouse spermatids (Schmidt et al., 2000). While the problem of Cre toxicity is known, it is largely ignored as appropriate controls are frequently missing. Highlighting this, Lee et al. (2006) did a meta-analysis of studies where the RIP-Cre mice had been used to probe pancreatic β-cell function. They found that the majority of studies had failed to investigate the impact of the Cre transgene relative to their wild-type littermates (Lee et al., 2006). A similar pattern emerges when the neuroscience literature is examined. Here we investigate the potential impact of Cre toxicity on seizure susceptibility in two conditional mice models, Emx1-Cre and Dlx5/6-cre. Emx1 encodes a transcription factor that regulates several embryonic processes in excitatory neurons including migration and differentiation (Gorski et al., 2002). Emx1-Cre mice have the Cre gene “knocked in” to the endogenous Emx1 locus resulting in a forebrain specific expression pattern in both neuron and astrocytes (Iwasato et al., 2000, Iwasato et al., 2004). The Dlx genes are also transcription factors that are required for the proper differentiation and migration of ventrally derived inhibitory interneurons (Batista-Brito et al., 2008). Dlx5/6-Cre mice are a transgenic strain that expresses Cre and EGFP in forebrain GABAergic neurons under the control of the Dlx5/6 enhancer element id6/id5 (Stenman et al., 2003). We demonstrate that Cre expression in both these conditional mouse models is sufficient to alter seizure susceptibility as assessed by a gold-standard proconvulsant test. These results demonstrate the potential for Cre-mediated toxicity to alter neuronal network function. This is a likely confound to the interpretation of cellular and behavior phenotypes in any loxP-target mice when using these conditional models. In general, these results strongly imply a need for appropriate negative controls when using Cre-based conditional models to rule out neurotoxicity.

Section snippets

Mice

All experiments were approved by the Animal Ethics Committee at the Florey Neuroscience Institutes. Experiments have been carried out in accordance with the EC directive for animal experimentation. Mice were housed at the Florey Neuroscience Institutes animal facility under a 12 h light/dark cycle with water and food freely available. Both male and female mice were used between the ages of P35 and P40. Emx1-Cre mice were obtained from Iwasato et al. (Iwasato et al., 2000), and backcrossed to

Results

Emx1-Cre mice were healthy, active and showed no obvious behavioral phenotypes including gait, socialization and aggression on handling. No differences in weight was observed between Emx1-Cre mice and their wild-type littermates (16.0 ± 1.0 g, n = 11 vs. 16.8 ± 0.8 g, n = 7, p = 0.7). scPTZ provides a measure of neuronal network excitability with increased sensitivity correlating with an epileptic phenotype (Tan et al., 2007). For both seizure endpoints latency was significantly shorter in the Emx1-Cre

Discussion

Conditional gene targeting is widely used in neuroscience probing genetic influences in a temporal and/or cell- specific manner (Gaveriaux-Ruff and Kieffer, 2007). PTZ-based proconvulsant method is commonly used to test compounds for antiepileptic activity (Loscher, 2011). The method is also commonly used to probe seizure susceptibility in genetic rodent models (Yang and Frankel, 2004). General behavior, health and gross brain morphology are normal in the two Cre models suggesting that major

Conclusion

Despite the potential drawbacks of toxicity associated with the Cre–lox system this gene technology remains a powerful tool with which to investigate neurobiological questions. Our data and that of others (for review see (Schmidt-Supprian and Rajewsky, 2007)) highlights the importance for appropriate control experiments that compare wild-type mice with those that only carry the Cre transgene.

Acknowledgments

Supported by NHMRC project grant 628520 to (CAR) and a NHMRC program grant 400121 to SP. CAR also acknowledge the support of an ARC Future Fellowship (FT0990628), The Florey Neuroscience Institutes. The Florey Neuroscience Institutes are supported by the Victorian Government.

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      In addition, Amigo2 has been localized only to CA2 pyramidal cells and hilar neurons (Hitti and Siegelbaum, 2014), which is a small fraction of neurons in the brain. However, it is notable that in studies of others, DLX-Cre+/−mice did appear to have more seizures than DLX-Cre−/− mice despite the absence of viral injection to experimentally manipulate cells expressing DLX (Kim et al., 2013). Other laboratories have used Cre+/− lines without viral expression in their research, however, and it has not been shown that hemizygous Cre on its own has major effects on endpoints we investigated, e.g., seizures after SE.

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