ABSTRACT
RNA interference (RNAi) can be induced in vitro either by application of synthetic short interfering RNAs (siRNAs), or by intracellular expression of siRNAs or short hairpin RNAs (shRNAs) from transfected vectors. The most widely used promoters for siRNA/shRNA expression are based on polymerase III (Pol III)-dependent transcription. We developed an alternative vector for siRNA/shRNA expression, using a mouse RNA polymerase I (Pol I) promoter. Pol I-dependent transcription serves in cells for production of ribosomal RNA (rRNA), and as such, is ubiquitously and stably active in different cell types. As Pol I-dependent transcription is highly species-specific, Pol I-based system provides an important biosafety advantage with respect to silencing of genes with unknown functions.
Subject(s)
Genetic Vectors , RNA Interference , RNA Polymerase I/metabolism , RNA, Small Interfering/metabolism , Animals , Cell Line , Genetic Vectors/chemistry , Humans , Mice , Promoter Regions, Genetic , RNA, Untranslated/genetics , RNA, Untranslated/metabolism , Species Specificity , Transcription, GeneticABSTRACT
CD81, also known as target of the antiproliferative antibody, is known to be expressed in astrocytes and involved in cell adhesion and, recently, we demonstrated its induction exclusively in the accumbens following cocaine. In the present study, the sensitivity of CD81-deficient mice to behavioral effects of cocaine was evaluated. It was found that CD81-deficient mice exhibited altered sensitivity to cocaine as assessed in the place preference conditioning paradigm and locomotor activity. This deficit in place preference conditioning was not accompanied by a deficit in acquisition or retention of water maze behavior. In addition, CD81 knockout mice exhibited higher levels of nucleus accumbens dopamine as compared to their controls. These observations are discussed in the context of the role of CD81 in cocaine-mediated behaviors.
Subject(s)
Antigens, CD/physiology , Cocaine/toxicity , Maze Learning/drug effects , Membrane Proteins , Motor Activity/drug effects , Nerve Tissue Proteins/physiology , Spatial Behavior/drug effects , Animals , Antigens, CD/genetics , Corpus Striatum/metabolism , Dopamine/metabolism , Drug Resistance , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/genetics , Neurotransmitter Agents/metabolism , Nucleus Accumbens/metabolism , Tetraspanin 28ABSTRACT
CD81, a tetraspanin transmembrane protein involved in cell adhesion, was found by differential display to be upregulated in the nucleus accumbens of rat brain following acute cocaine treatment (four injections of 30 mg/kg every 2 h followed by 24 h withdrawal). Cocaine-induced expression of CD81 in adult rat brain was confirmed by quantitative real-time RT-PCR. Its expression in neurons and its function in the brain are unknown. In situ hybridization shows a neuron-specific expression pattern in brain regions functionally related to the regulation of cardiovascular function and fluid homeostasis. CD81 displays codistribution to galanin and, to a lesser extent, to vasopressin. These findings add to data that suggest a connection between the brain reward pathway and the centers regulating endocrine and autonomic functions, in relation to neurochemical, behavioral, and somatic consequences of drug abuse.
