The genetic manipulation of target genes in transgenic animals in vivo is one of the most valuable tools for assessing gene functions and for modelling human diseases. The most popular approaches for the generation of genetically modified mice are the targeted engineering of genomic DNA in embryonic stem cells to introduce gene knockouts and the DNA microinjection into the pronuclei of fertilized eggs, which results in random integration of the transgene into the genome for the overexpression of gene products.
These valuable techniques have inherent limitations related to the irreversibility and ubiquity of the germ line genetic modifications. If essential genes are targeted, this will potentially lead to embryonic lethality or activation of compensatory mechanisms, which complicates or even impedes the phenotypic analyses of these animal models [1
]. In particular this holds true for the large number of genes involved in the formation of neuronal structures emerging late in embryonic development or at early postnatal stages. To overcome these limitations, several techniques for the spatial and temporal control of gene expression in genetically modified mice have been proposed and developed [2
], of which the Cre/loxP recombination system [3
] and the tet-controlled transcription activation system (Tet system [4
]) have become the most widely applied and characterized [1
Cre is a site-specific recombinase that catalyses recombination between its recognition sites, loxP, leading to an inversion or deletion of a loxP-flanked DNA sequence. Hence, Cre recombinase can be applied to delete genes or to activate transcription by removing a transcriptional termination (STOP) sequence [1
]. To obtain an inducible version of Cre, the recombinase was fused to a mutant form of the human oestrogen receptor, leading to cytoplasmic localization and therefore inactivation of the fusion protein (CreERT2) [5
]. Once the ligand tamoxifen is applied, CreERT2 translocates into the nucleus, where recombination takes place.
The Tet system is based on two central elements, the tetracycline (tet)-controlled transactivator (tTA) and a specific responsive promoter (Ptet), which controls expression of the transgene [4
]. Ptet is specifically activated by binding of tTA. Tet and tet derivatives such as doxycycline hydrochloride (Dox) interfere with the DNA-binding activity of tTA, thereby abolishing transcriptional activation of Ptet. By tissue-specific expression of Cre recombinase or tTA, the impact of transgenic perturbation can be limited to defined cell populations.
For many scientific questions the rat is the preferential animal model [6
]. This is mainly related to its larger body size, relevance to human physiology and large body of experimental experience with these animals [7
]. Rats are of particular importance in neuroscience because they are the preferred species for behavioural testing of higher cognitive functions, multielectrode recordings, studies of neuronal regeneration [8
] and for gene therapy experiments [9
Up to now, the establishment of techniques for the conditional manipulation of genes in the rat is far behind those for mice [10
]. Furthermore, in light of the recent technological breakthroughs that allow targeted genomic manipulations in rats, including the application of zinc finger or transcription activator-like effector nucleases [11
] and the development of germ line-competent rat embryonic stem cells [14
], there is an urgent need to introduce conditional technologies for rats and to develop specific Cre driver lines to realize tissue-specific genetic modifications.
In this report, we describe the generation of transgenic rat lines expressing tTA and the tamoxifen-inducible Cre recombinase CreERT2 under the control of the forebrain-specific Ca2+/calmodulin-dependent protein kinase IIa (CaMKIIα) promoter. For functional characterization of the CaMKIIα-tTA and CaMKIIα-CreERT2 lines, we generated novel tTA and Cre reporter lines that allow faithful monitoring of spatial and temporal Ptet-controlled and Cre/loxP-mediated gene regulation. These novel tTA and CreERT2 lines can be used for inducible transgene overexpression, as exemplified herein with reporter genes, or for inducible gene knockouts once loxP-flanked gene alleles become available for the rat genome.