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We previously generated a knock-in mouse line with a cocaine-insensitive dopamine transporter (DAT-CI mice). These mice lost several behavioral responses to cocaine, but retained their response to amphetamine. DAT-CI mice are hyperdopaminergic due to reduced DAT function, and may thus be a good model for studying attention deficit hyperactivity disorder (ADHD). These mice had been behaviorally characterized while they were on a mixed genetic background. However as the colony was propagated over time, the mixed genetics were shifted toward a pure C57Bl/6J background--via a common breeding scheme known as "backcrossing." Several phenotypes appeared to have changed during this time frame. In this study, we investigated whether backcrossing altered the hyperlocomotive phenotype and behavioral responses to amphetamine, a drug used to treat ADHD. C57-congenic DAT-CI mice had high spontaneous locomotor activity that could be suppressed by low doses of amphetamine. Furthermore, their locomotion was not stimulated by very high doses of amphetamine (20mg/kg). After the reversion to a mixed genetic background by breeding with the 129 strain, the C57:129 hybrid DAT-CI mice displayed reduced basal locomotor activity compared to the C57-congenic mutant mice, and regained locomotor stimulation by high-dose amphetamine. The calming effect of amphetamine at low doses was retained in both strains. In summary, reduced DAT function in DAT-CI mice leads to a hyperdopaminergic state, and an ADHD-like phenotype in both strains. The data show that the genetic background of DAT-CI mice affects their locomotor phenotypes and their responses to amphetamine. Since the differences in genetic background between the strains of mice have a significant impact on the ADHD-like phenotype and the response to amphetamine, further study with these strains could identify the genetic underpinnings affecting the severity of ADHD-related symptoms and the treatment response., (Copyright © 2012 Elsevier Inc. All rights reserved.)

RNA interference is a cellular mechanism regulating levels of mRNAs. It has been widely exploited to knock down specific protein targets. The selected interfering RNA sequence greatly influences its ability to knock down the target. Here we present a method for constructing multiple testing plasmids which express small hairpin RNAs (shRNA) targeting different regions of an mRNA. A simple fluorescence test in cultured cells allows convenient evaluation of mRNA knockdown by many different shRNAs on 96-well plates. We show that software predicted shRNAs have varying efficacies and only 2 of the 7 tested shRNAs significantly knocked down their targets., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Serotonin transporter (SERT) is one of the key protein targets of cocaine. Despite intensive studies, it is not clear where cocaine binds to its targets and what residues are involved in cocaine binding. We have cloned the serotonin transporter from silkworm (Bombyx mori, bmSERT). When expressed in cultured cells, bmSERT is over 20-fold less sensitive to cocaine than Drosophila melanogaster SERT (dmSERT). We performed species-scanning mutagenesis using bmSERT and dmSERT. There are two adjacent threonine residues in transmembrane domain 12 of bmSERT where the corresponding residues are two serines in dmSERT and in all known mammalian monoamine transporters. Replacing the serine residues with threonines in dmSERT reduces cocaine sensitivity; while switching the two threonine residues in bmSERT to serines increased cocaine sensitivity. Mutations at the corresponding residues in dopamine transporter also changed cocaine affinity. Our results suggest that the conserved serine residues in SERT contribute to high-affinity cocaine binding.