Abstract
Short cell-type specific promoter sequences are important for targeted gene therapy and studies of brain circuitry. We report on the ability of short promoter sequences to drive fluorescent protein expression in specific types of mammalian cortical inhibitory neurons using adeno-associated virus (AAV) and lentivirus (LV) vectors. We tested many gene regulatory sequences derived from fugu (Takifugu rubripes), mouse, human, and synthetic composite regulatory elements. All fugu compact promoters expressed in mouse cortex, with only the somatostatin (SST) and the neuropeptide Y (NPY) promoters largely restricting expression to GABAergic neurons. However these promoters did not control expression in inhibitory cells in a subtype specific manner. We also tested mammalian promoter sequences derived from genes putatively coexpressed or coregulated within three major inhibitory interneuron classes (PV, SST, VIP). In contrast to the fugu promoters, many of the mammalian sequences failed to express, and only the promoter from gene A930038C07Rik conferred restricted expression, although as in the case of the fugu sequences, this too was not inhibitory neuron subtype specific. Lastly and more promisingly, a synthetic sequence consisting of a composite regulatory element assembled with PAX6 E1.1 binding sites, NRSE and a minimal CMV promoter showed markedly restricted expression to a small subset of mostly inhibitory neurons, but whose commonalities are unknown.
Highlights
The mammalian cerebral cortex contains two major classes of neurons, excitatory and inhibitory
We previously reported that rAAV2/1 transduces cortical inhibitory neurons more efficiently than excitatory neurons, while VSV-G-LV transduces excitatory neurons more efficiently than inhibitory neurons (Nathanson et al, 2009)
To target specific neuron cell-types in mammals, we tested regulatory sequences derived from multiple sources: (i) promoter sequences derived from putative orthologous fugu genes, (ii) mammalian promoter segments from genes putatively co-expressed in known inhibitory neuron subtypes, and (iii) assembled composite regulatory elements (CREs) containing transcription factor binding sites (TFBSs)
Summary
The mammalian cerebral cortex contains two major classes of neurons, excitatory and inhibitory. Genetic methods for selective neuronal tracing or inactivation, for which viruses are the best delivery vehicles, have been developed to directly study particular cells, but the utility of these genetic tools requires the ability to selectively target gene expression to specific cell-types (Callaway, 2005; Luo et al, 2008) One such way is to use short cis-regulatory sequences (termed promoters), which can regulate gene expression using enhancers, silencers, and/or insulator/boundary elements. We tested putative regulatory regions from fugu genes orthologous to mammalian inhibitory neuron specific markers PV, CR, SST and NPY. These coexpressed genes provided additional candidates for targeting inhibitory neuron subtypes Motivated by this data, we chose the fugu genes titin-cap (TCAP) and secreted frizzled-related protein 2 (SFRP2), which respectively express in PV and SST cell-types in the mouse. We created CREs by combining DR1 (Okuno et al, 2001) or E1.1 (Scardigli et al, 2003) motifs with a neuron restrictive silencing element (NRSE) (Bessis et al, 1997; Millecamps et al, 1999) and a minimal cytomegalovirus (mCMV) promoter
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