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1.  Adenoviral vectors for highly selective gene expression in central serotonergic neurons reveal quantal characteristics of serotonin release in the rat brain 
BMC Biotechnology  2009;9:23.
5-hydroxytryptamine (5 HT, serotonin) is one of the key neuromodulators in mammalian brain, but many fundamental properties of serotonergic neurones and 5 HT release remain unknown. The objective of this study was to generate an adenoviral vector system for selective targeting of serotonergic neurones and apply it to study quantal characteristics of 5 HT release in the rat brain.
We have generated adenoviral vectors which incorporate a 3.6 kb fragment of the rat tryptophan hydroxylase-2 (TPH-2) gene which selectively (97% co-localisation with TPH-2) target raphe serotonergic neurones. In order to enhance the level of expression a two-step transcriptional amplification strategy was employed. This allowed direct visualization of serotonergic neurones by EGFP fluorescence. Using these vectors we have performed initial characterization of EGFP-expressing serotonergic neurones in rat organotypic brain slice cultures. Fluorescent serotonergic neurones were identified and studied using patch clamp and confocal Ca2+ imaging and had features consistent with those previously reported using post-hoc identification approaches. Fine processes of serotonergic neurones could also be visualized in un-fixed tissue and morphometric analysis suggested two putative types of axonal varicosities. We used micro-amperometry to analyse the quantal characteristics of 5 HT release and found that central 5 HT exocytosis occurs predominantly in quanta of ~28000 molecules from varicosities and ~34000 molecules from cell bodies. In addition, in somata, we observed a minority of large release events discharging on average ~800000 molecules.
For the first time quantal release of 5 HT from somato-dendritic compartments and axonal varicosities in mammalian brain has been demonstrated directly and characterised. Release from somato-dendritic and axonal compartments might have different physiological functions. Novel vectors generated in this study open a host of new experimental opportunities and will greatly facilitate further studies of the central serotonergic system.
PMCID: PMC2672940  PMID: 19298646
2.  Viral vectors based on bidirectional cell-specific mammalian promoters and transcriptional amplification strategy for use in vitro and in vivo 
BMC Biotechnology  2008;8:49.
Using cell-type-specific promoters to restrict gene expression to particular cells is an attractive approach for gene therapy, but often hampered by insufficient transcriptional activity of these promoters. Previous studies have shown that transcriptional amplification strategy (TAS) can be used to enhance the activity of such promoters without loss of cell type specificity. Originally TAS involved the use of two copies of a cell-specific promoter leading to generation of large expression cassettes, which can be hard to use given the space limitations of the conventional viral gene expression vectors.
We have now developed a new bidirectional lentiviral vector system, based on TAS that can enhance the transcriptional activity of human synapsin-1 (SYN) promoter and the compact glial fibrillary acidic protein (GfaABC1D) promoter. In the opposite orientation, a minimal core promoter (65 bp) derived from the human cytomegalovirus (CMV) was joined upstream of the SYN promoter or GfaABC1D promoter. This led to the formation of synthetic bidirectional promoters which were flanked with two gene expression cassettes. The 5' cassette transcribed the artificial transcriptional activator. The downstream cassette drove the synthesis of the gene of interest. Studies in both cell cultures and in vivo showed that the new bidirectional promoters greatly increased the expression level of the reporter gene. In vivo studies also showed that transgene expression was enhanced without loss of cell specificity of both SYN and GfaABC1D promoters.
This work establishes a novel approach for creating compact TAS-amplified cell-specific promoters, a feature important for their use in viral backbones. This improved approach should prove useful for the development of powerful gene expression systems based on weak cell-specific promoters.
PMCID: PMC2396617  PMID: 18485188
3.  Single fluorescent protein-based Ca2+ sensors with increased dynamic range 
BMC Biotechnology  2007;7:37.
Genetically encoded sensors developed on the basis of green fluorescent protein (GFP)-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca2+ sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer) changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP).
Here we report significant progress on the development of the latter type of Ca2+ sensors. Derived from the knowledge of previously reported cpFP-based sensors, we generated a set of cpFP-based indicators with different spectral properties and fluorescent responses to changes in Ca2+ concentration. Two variants, named Case12 and Case16, were characterized by particular high brightness and superior dynamic range, up to 12-fold and 16.5-fold increase in green fluorescence between Ca2+-free and Ca2+-saturated forms. We demonstrated the high potential of these sensors on various examples, including monitoring of Ca2+ response to a prolonged glutamate treatment in cortical neurons.
We believe that expanded dynamic range, high brightness and relatively high pH-stability should make Case12 and Case16 popular research tools both in scientific studies and high throughput screening assays.
PMCID: PMC1931437  PMID: 17603870

Results 1-3 (3)