The neurotransmitter serotonin (5-HT) is produced by a two step enzymatic process from the amino acid tryptophan, primarily by neurons located in the raphe nuclei. These neurons project too many regions of the brain including the cortex, thalamus, hypothalamus, and amygdala. Released serotonin can act on over 14 different receptors, expressed by both presynaptic and postsynaptic neurons. Following vesicular release, the action of 5-HT is terminated by its removal from the synapse by the serotonin transporter (5-HTT, SERT, SLC6A4). Deregulation of serotonergic pathways has been implicated in many psychiatric and affective disorders including depression, autism, and obsessive compulsive disorder. These associations are supported by the demonstration that 5-HTT is the major site of action of many antidepressants. 5-HTT inhibitors (SRIs) are effective, not only in treating depression, but also in the treatment of other disorders including anxiety, obsessive compulsive disorder (OCD), and some aspects of autism. Genetic studies have reported association of both common and rare polymorphisms in the 5-HTT gene with increased risk for psychiatric and affective disorders. These results not only provide a rationale for targeting 5-HTT in the treatment of these illnesses, but also suggest that altered expression and activity of the transporter may contribute to the development of the illness itself. It has proved difficult to directly test either the contribution of polymorphisms to disturbances in serotonin homeostasis in vivo or the ability of these disturbances to manifest as measurable changes in behavior. To begin to address these questions, we propose the generation of novel mouse lines. These lines are designed to test the hypothesis that polymorphisms in the 5-HTT promoter alter expression of this gene and that the resulting changes in serotonin metabolism are sufficient to cause measurable changes in behavior. To accomplish this goal we have developed a unique two step method for exchange of the locus encoding a specific mouse gene with its human counterpart. Because the entire mouse gene, including the promoter and 5'and 3'flanking regions, is exchanged with the corresponding human sequence, it is possible using this method to address not only the functionality of non-synonymous SNPs but also non- coding polymorphisms in introns and regulatory regions of the gene. Using this vector system we propose to generate mice in which the endogenous 5-Htt gene is excised and replaced with a human 5-HTT gene with either the long (LA), the long (LG), or the short (S) allele of the 5-HTT linked polymorphic region (HTTLPR). The expression of 5-HTT, the activity of the serotonin pathway, and the response of the mice in behavioral tests will provide information concerning the functionality of the three promoter polymorphism. The replacement of the entire mouse gene with the syntenic human DNA will not only facilitate future testing of additional 5-HTT SNPs and variants, but also simplify the evaluation of gene-gene interactions by the intercross of these mouse lines to lines expressing disease related polymorphisms at other loci. Genetic studies are identifying an ever increasing number of polymorphisms linked to risk for affective and psychiatric illness. Determining the functionality of these genetic variations has proved difficult, particularly when no change in the primary structure of the encoded protein is predicted by the polymorphism. In this application, we present the development of a new strategy for the exchange of mouse genes with their human orthologues. We show that after this exchange SNPs can be introduced into the human DNA segment of the mouse genome. As this genetic manipulation is carried out in mouse embryonic stem cells, it is possible to generate mouse lines that differ only in a particular polymorphism associated with risk for disease in the human population. These mice can be used to study the impact of this polymorphism on gene expression, protein function, development, and on the behavioral tests/models known to be sensitive to alterations in this specific pathway in rodents.