Viral vector-mediated Cre recombinase expression in substantia nigra induces lesions of the nigrostriatal pathway associated with perturbations of dopamine-related behaviors and hallmarks of programmed cell death.

Cre/loxP recombination is a widely used approach to study gene function in vivo, using mice models expressing the Cre recombinase under the control of specific promoters or through viral delivery of Cre-expressing constructs. A profuse literature on transgenic mouse lines points out the deleterious effects of Cre expression in various cell types and tissues, presumably by acting on illegitimate loxP-like sites present in the genome. However, most studies reporting the consequences of Cre-lox gene invalidation often omit adequate controls to exclude the potential toxic effects of Cre, compromising the interpretation of data. In this study, we report the anatomical, neurochemical, and behavioral consequences in mice of adeno-associated virus (AAV)-mediated Cre expression in the dopaminergic nuclei substantia nigra, at commonly used viral titers (3 × 109 genome copies/0.3 µL or 2 × 109 genome copies/0.6 µL). We found that injecting AAV-eGFP-Cre into the SN engendered drastic and reproducible modifications of behavior, with increased basal locomotor activity as well as impaired locomotor response to cocaine compared to AAV-eGFP-injected controls. Cre expression in the SN induced a massive decrease in neuronal populations of both pars compacta and pars reticulata and dopamine depletion in the nigrostriatal pathway. This anatomical injury was associated with typical features of programmed cell death, including an increase in DNA break markers, evidence of apoptosis, and disrupted macroautophagy. These observations underscore the need for careful control of Cre toxicity in the brain and the reassessment of previous studies. In addition, our findings suggest that Cre-mediated ablation may constitute an efficient tool to explore the function of specific cell populations and areas in the brain, and the impact of neurodegeneration in these populations.

Cre recombinase expression or topical tamoxifen treatment do not affect retinal structure and function, neuronal vulnerability or glial reactivity in the mouse eye.

Mice with a constitutive or tamoxifen-induced Cre recombinase (Cre) expression are frequently used research tools to allow the conditional deletion of target genes via the Cre-loxP system. Here we analyzed for the first time in a comprehensive and comparative way, whether retinal Cre expression or topical tamoxifen treatment itself would cause structural or functional changes, including changes in the expression profiles of molecular markers, glial reactivity and photoreceptor vulnerability. To this end, we characterized the transgenic α-Cre, Lmop-Cre and the tamoxifen-inducible CAGG-CreER™ mouse lines, all having robust Cre expression in the neuronal retina. In addition, we characterized the effects of topical tamoxifen treatment itself in wildtype mice. We performed morphometric analyses, immunohistochemical staining, in vivo ERG and angiography analyses and realtime RT-PCR analyses. Furthermore, the influence of Cre recombinase or topical tamoxifen exposure on neuronal vulnerability was studied by using light damage as a model for photoreceptor degeneration. Taken together, neither the expression of Cre, nor topical tamoxifen treatment caused detectable changes in retinal structure and function, the expression profiles of investigated molecular markers, glial reactivity and photoreceptor vulnerability. We conclude that the Cre-loxP system and its induction through tamoxifen is a safe and reliable method to delete desired target genes in the neural retina.

[Issues and solutions of conditional gene targeting].

Conditional gene targeting, based on the site-specific recombination system such as Cre-loxP, plays a vital role in the study of gene functions and the generation of disease mouse models. It was always under consideration that there were problems in the Cre-loxP recombination system, such as illegal expression pattern of Cre transgene, variation of Cre recombination efficiency and toxicity of Cre recombinase, as well as the potential influences of genetic background, breeding strategy, experimental control and gene compensation. Oversights of these issues may have a profound influence on the accuracy of gene functional dissection and conditional gene targeting mice phenotypic interpretation. Accordingly, solutions should be adopted including delicate regulative control of temporal-spatial specific Cre expression, detailed detection of Cre recombination efficiency, reduction of Cre toxicity, simplification of mouse genetic background, optimization of breeding, setting up of proper control and combined conditional gene targeting.

Direct hematological toxicity and illegitimate chromosomal recombination caused by the systemic activation of CreERT2.

The CreER(T2) for conditional gene inactivation has become increasingly used in reverse mouse genetics, which enables temporal regulation of Cre activity using a mutant estrogen binding domain (ER(T2)) to keep Cre inactive until the administration of tamoxifen. In this study, we present the severe toxicity of ubiquitously expressed CreER(T2) in adult mice and embryos. The toxicity of Cre recombinase or CreER(T2) in vitro or in vivo organisms are still less sufficiently recognized considering the common use of Cre/loxP system, though the toxicity might compromise the phenotypic analysis of the gene of interest. We analyzed two independent lines in which CreER(T2) is knocked-in into the Rosa26 locus (R26CreER(T2) mice), and both lines showed thymus atrophy, severe anemia, and illegitimate chromosomal rearrangement in hematopoietic cells after the administration of tamoxifen, and demonstrated complete recovery of hematological toxicity in adult mice. In the hematopoietic tissues in R26CreER(T2) mice, reduced proliferation and increased apoptosis was observed after the administration of tamoxifen. Flow cytometric analysis revealed that CreER(T2) toxicity affected several hematopoietic lineages, and that immature cells in these lineages tend to be more sensitive to the toxicity. In vitro culturing of hematopoietic cells from these mice further demonstrated the direct toxicity of CreER(T2) on growth and differentiation of hematopoietic cells. We further demonstrated the cleavage of the putative cryptic/pseudo loxP site in the genome after the activation of CreER(T2) in vivo. We discussed how to avoid the misinterpretation of the experimental results from potential toxic effects due to the activated CreER(T2).

Reduction of Cre recombinase toxicity in proliferating Drosophila cells by estrogen-dependent activity regulation.

The Cre/loxP site-specific recombination system has been used successfully for genome manipulation in a wide range of species. However, in Drosophila melanogaster, a major model organism for genetic analyses, the alternative FLP/FRT system, which is less efficient at least in mammalian cells, has been established, primarily for the generation of genetic mosaics for clonal analyses. To extend genetic methodology in D. melanogaster, we have created transgenic lines allowing tissue-specific expression of Cre recombinase with the UAS/GAL4 system. Surprisingly, chronic expression of Cre recombinase from these transgenes (UAST-cre) was found to be toxic for proliferating cells. Therefore, we also generated transgenic lines allowing the expression of Cre recombinase fused to the ligand-binding domain of the human estrogen receptor (UASP-cre-EBD). We demonstrate that recombination can be efficiently dissociated from toxicity by estrogen-dependent regulation of recombinase activity of the UASP-cre-EBD transgene products.

Self-excising retroviral vectors encoding the Cre recombinase overcome Cre-mediated cellular toxicity.

The Cre-lox system is often used to manipulate sequences in mammalian genomes. We have observed that continuous expression of the Cre recombinase in cultured cells lacking exogenous lox sites caused decreased growth, cytopathic effects, and chromosomal aberrations. Cre mutants defective in DNA cleavage were not geno- or cytotoxic. A self-excising retroviral vector that incorporates a negative feedback loop to limit the duration and intensity of Cre expression avoided measurable toxicity, retained the ability to excise a target sequence flanked by lox sites, and may provide the basis of a less toxic strategy for the use of Cre or similar recombinases.