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  • In our study we evaluated adenovirus


    In our study we evaluated adenovirus type 5 (Ad5) as a vehicle for delivering Cre recombinase in conditional transgenic mouse based on the Cre-loxP system, in order to achieve GlyT1 gene modulation in a specific thalamic region in the adult animal. GlyT1 is a sodium- and chloride-coupled glycine transporter that mediates the uptake of glycine from the extracellular space into the cytosol (Aragón and López-Corcuera, 2003). Glycine exerts several functional roles in the mammalian CNS including that as an inhibitory neurotransmitter, in particular in the caudal regions of the adult anderson kpt (Eulenburg et al., 2005), and as an obligatory co-agonist at the N-methyl-d-aspartate (NMDA) receptor (Johnson and Ascher, 1987). In mammalian brain, two high-affinity neuron and glial plasma membrane-located transporters, GlyT1 and GlyT2, have been cloned. The GlyT1 gene is widely expressed throughout the brain with highest levels of expression in caudal areas and with cellular localization in both membrane and processes of glial cells, mainly astrocytes (Aragón and López-Corcuera, 2003). In the forebrain, GlyT1 mRNA has been detected in the cortex, the hippocampus, the hypothalamus and in all thalamic regions (Gabernet et al., 2005). Modulation of glycine transporter activity represents a promising approach for the treatment of several disorders and in particular targeting the inhibition of GlyT1 as a strategy to address NMDA receptor hypofunction in schizophrenia has attracted considerable interest (Yang and Svensson, 2008). Recently, Eulenburg and colleagues reported the use of a conventional germline deletion approach to generate mice exhibiting a disruption in the GlyT1 gene in their glial cells (GlyT1(+)/(+)/GFAP-Cre) (Eulenburg et al., 2010). This was achieved by crossing the GlyT1-targeted mice with mice expressing Cre under the control of the glial-specific GFAP promoter. The majority of these GlyT1-disrupted mice died prematurely and there was some variability between the mice in the timing of death. The strategy used by Eulenburg and colleagues allows the phenotypic analysis of GlyT1 deletion in all glial cells, however, the strategy does have its limitations. First of all, since deletion occurred during fetal development, genetic compensation and developmental adaptations could explain the phenotypic variations between mice. In addition, such a strategy does not allow investigation of GlyT1 deletion in a precise spatio-temporal manner in young or adult mice, as we have demonstrated specifically in the thalamus.
    Materials and methods
    Discussion The development of the Cre-loxP system has represented an important achievement to enable gene manipulation. Cre recombinase catalyzes site-specific recombination of the DNA region located between the loxP sites, thus, mediating the inactivation or induction of the target gene in the presence of Cre protein. Viral vectors have been used in association with the Cre-loxP system, to generate inducible transgenic models that allow flexible temporal–spatial control of gene modulation. This can be particularly useful in the field of neurobiological research where there may be a requirement to dissect the role of a target gene only in the brain or in a particular brain region, when the target gene is expressed in different tissues with different functional roles. We investigated the use of an adenoviral vector to deliver Cre recombinase and mediate the suppression of GlyT1, a therapeutic target for the treatment of schizophrenia, in the thalamic nuclei of adult transgenic mice. We employed Ad5-derived vectors since they have been reported to mediate Cre expression and loxP-dependent recombination in rodent brain (Wang et al., 1996, Thévenot et al., 2003). Moreover, unlike adeno-associated virus vectors (AAV), Ad5-derived vectors can efficiently infect both dividing and non-dividing cells in vitro and have been reported to infect preferentially glia (around 60%) and in lower level neurons (∼30%) in rodent brain (Kremer, 2005). Since the target of our study is the glycine trasporter GlyT1, a protein expressed predominantly in glial cells (Aragón and López-Corcuera, 2003) we employed an Ad5-based Cre-expression vector to mediate site-specific recombination and suppression of GlyT1 expression in the glial cells of conditional GlyT1 mice. As we demonstrated in our study, this technology allows the induction, as seen for Rosa26 mice, or suppression, as seen in the cGlyT1 model, of the target gene in an extremely restricted brain region, specifically only in the injected thalamic nuclei. Moreover, another advantage of this methodology is the possibility to use the same viral vector and transgenic animal model to investigate the effect of target gene modulation in several distinct brain regions. This is of particular relevance in our study since the pattern of expression of GlyT1 is widespread in the brain and GlyT1 protein is involved in different functional roles in distinct neuronal populations (Aragón and López-Corcuera, 2003).