A silencer or bad regulatory element that would normally suppress TH expression might be missing from your relatively small constructs, as has been proposed to be a potential reason for ectopic transgene expression in the mouse and in the zebrafish [28,29]. performed on individual GFP+ cells collected from dispersed retinal cell cultures for and dopamine transporter (promoter-driven GFP exhibited strong expression in the brain and retina during zebrafish development. In juvenile and adult fish retinas, GFP was expressed in cells located in the inner nuclear layer. Immunocytochemistry with antibodies for GFP and TH showed that 292% of GFP-labeled cells also expressed TH. Two subpopulations of GFP-labeled cells were recognized by fluorescent microscopy: bright GFP-expressing cells and dim GFP-expressing cells. Seminested single-cell RTCPCR showed that 71% of dim GFP-expressing cells expressed both and mRNA. Loose-patch voltage-clamp recording from dim GFP-labeled cells in retinal whole mounts revealed that many of these dopaminergic neurons are spontaneously active in darkness. Conclusions Although this collection is not a completely specific reporter for dopaminergic neurons, using relative GFP intensity we are able to enrich for the selection of retinal dopaminergic cells in vitro and in situ in molecular and electrophysiological experiments. This transgenic collection provides a useful tool for studying retinal dopaminergic cells in the zebrafish. Introduction In the central nervous system, dopamine (DA) plays important functions in modulating a variety of physiologic events such Veralipride as movement, emotion, memory, and reward processing. In the vertebrate retina, dopamine is usually Veralipride involved in mediating neuronal adaptation to light [1,2], and circadian rhythmicity [3-5], as well as cell survival and vision growth [6,7]. In teleost retinas, dopamine is usually released by dopaminergic interplexiform cells (DA-IPCs), which contact horizontal and bipolar cell dendrites in the outer plexiform layer (OPL), and receive input from amacrine and bipolar cell terminals in the inner retina [1,8-10]. DA-IPCs have been proposed to be a centrifugal pathway for information flow from your inner to the outer retina [9,11], and they have been shown to mediate the modulatory effect of olfactory input on retinal ganglion cell activity [12]. Despite the diverse functions of DA cells in retinal functions, the understanding of DA cell function has been limited because they have a low density in the retina and cannot be recognized in living retina by morphological characteristics [13,14]. In the mouse, transgenic lines have been created, in which reporter genes are driven by the promoter for the tyrosine hydroxylase (promoter. Here, we statement morphological, molecular, and physiologic characterization of the genetically labeled neurons in this transgenic zebrafish collection. Methods Transgenic zebrafish To generate the transgenic zebrafish, we isolated a genomic P1-derived artificial chromosome (PAC) clone, BUSMP706E03252Q3, made up of the zebrafish tyrosine hydroxylase 1 (cDNA sequence. To identify genomic fragments made up of the promoter region, we digested the PAC clone with several restriction enzymes and performed duplicate Southern hybridization using two probes. The first probe was a genomic PCR product of the 5UTR region of the locus. Following identification of the 5UTR by RACE, we designed two primers as shown in Table 1 for the genomic PCR. The second probe was generated from a previously published partial cDNA clone [17] by PvuII-BglII digest and contained approximately 100 bp of carboxyterminal portion of the coding region of the gene. Veralipride To identify a genomic fragment, which contains mostly the promoter region and not the coding region, we sought to isolate PAC restriction Veralipride fragments, based on Southern analysis, that are positive for the 5UTR probe but are unfavorable for the carboxyterminal probe. A XbaI-XhoI fragment was recognized, which fulfilled this criterion. The XbaI-XhoI fragment was further digested with EcoRI (EcoRI restriction site is found immediately downstream of the Th1 start ATG) and XhoI, and was cloned into a pBluescript II vector. The end sequencing of this fragment using T7 primer revealed that this fragment contains the genomic region of chromosome 25 starting at position 20376290 (Ensembl Zv7 assembly). Since the transcript begins at Rabbit Polyclonal to WAVE1 (phospho-Tyr125) the position 20364304, and since is usually oriented in reverse direction on this chromosome, the EcoRI-XhoI fragment encompasses 11986 bp of genomic promoter region; therefore, we refer to this fragment as 12 kb promoter fragment. The MmGFP chromophore coding region and SV40 polyA sequence was PCR-amplified from pG1 vector (gift of Chi-Bin Chien and Darren Gilmour, University or college of Utah, Salt Lake City, UT and EMBL) and cloned downstream of the 12 kb promoter fragment in the pBluescript II vector. To increase the translational efficiency, we adjusted the translational initiation context to better match the consensus Kozak sequence (GCCATGG). Transgenic zebrafish were generated by microinjection of 1C2 nl of 50 ng/l linearized DNA into 1- to 2-cell stage embryos (University or college of Freiburg, Freiburg, Germany). The founders were crossed with wild-type fish and their progeny were screened to establish transgenic lines. The transgene integration used for this.