Whole‐mount in situ hybridization with a tyrosine hydroxylase (th) (A–F) or a dopamine transporter (dat) cRNA probe (G–L). Amsterdam, A., S. Lin & N. Hopkins, 1995. Dev Dyn. Whole‐mount in situ hybridizations of tyrosine hydroxylase (th) and dopamine transporter (dat) in zebrafish embryos and larvae. (2001). I. To screen for clones containing the dopamine transporter (dat) gene from a zebrafish genomic PAC library (P1 artificial chromosome; RZPD, Berlin, Germany), a partial sequence of exon 1 of zebrafish dat gene was amplified and radioactively labeled by polymerase chain reaction (PCR), and later used as a probe for DNA hybridization. 8F–G′″). Here, we re‐examined this issue by observing MPTP‐treated Tg(dat:EGFP) embryos alive under a fluorescence microscope. A 3D Searchable Database of Transgenic Zebrafish Gal4 and Cre Lines for Functional Neuroanatomy Studies. Double fluorescent in situ hybridization with GFP and dat cRNA probes indicates that the transgene is expressed as predicted in dat‐expressing cells, notably in the ventral diencephalon (Fig. The 5 dpf Tg(dat:EGFP) embryos were horizontally cryosectioned and stained with anti‐GFP and anti‐TH antibodies. After 3× washes in 1×PBS for 10 min, the sections were preincubated with a blocking solution containing 10% new calf serum and 0.1% Tween 20 for 2 hr at room temperature, and then incubated with the same blocking solution containing the primary antibodies (see below) overnight in a humid chamber at 4°C. Visualization and experimental analysis of blood vessel formation using transgenic zebrafish. . For this application note, a fast protocol to place one single green fluorescent zebrafish larva in a suitable position for imaging in each well of a 96-well plate was developed. Developmental neurotoxic effects of graphene oxide exposure in zebrafish larvae (Danio rerio). 1C), although there are also a few cells that express GFP mRNA but not dat (Fig. EGFP (in green) was inserted in frame at the beginning of exon 1. In the present study, plasmid constructs containing green fluorescent protein (GFP) and the promoter of tyrosine hydroxylase (TH), a key synthetic enzyme for catecholamines, were produced. Conflicting results were obtained from previous studies with respect to the effects of MPTP on DA neuron survival in zebrafish (Bretaud et al., 2004; Lam et al., 2005; McKinley et al., 2005; Wen et al., 2008; Sallinen et al., 2009). Transgenesis is widely used in zebrafish due to its transparent and externally developing embryos (Lin, 2000). rapid linker-mediated PCR approach and subsequently ligated to a modified green fluorescent protein (gfp) reporter gene. COVID-19 is an emerging, rapidly evolving situation. All sections are shown as dorsal to the top. The embryonic zebrafish is a nearly ideal model system in which to use time-lapse imaging to study the development of the vertebrate nervous system in vivo. I: Projection of confocal images A–H. NLM 2002 Mar;223(2):204-15. doi: 10.1002/dvdy.10051. All sections are shown as anterior to the top. More than 90% of embryos injected with this construct in the presence of tol2 transposase mRNA showed GFP expression in areas located roughly between the eyes at 2 days post‐fertilization (dpf). Bruce Draper Zebrafish embryos develop rapidly. MPTP treatment caused the death of a significant albeit modest number of GFP‐positive DA neurons in the vDC of these fish. Research output: … 8, 2001, p. 845-849. A composite of different fluorescent focal planes was generated using the Image Pro software and merged with bright field image. The total size of the inserted DNA is 27 kb, containing the whole dat genomic sequence except for the last coding exon and 3′‐flanking region. This was accomplished by fusing GFP sequences to Islet-1 promoter/enhancer sequences that were sufficient for neural-specific expression. A th antisense probe was used as previously described (Xi et al., 2010). Huaihe Hosiptal, Henan University, Kaifeng, 475001 China. Transgenic zebrafish are a common vertebrate model system for the study of addictive behavior. The hyaloid vasculature in the zebrafish retina develops a barrier function at 3 dpf, which endows the zebrafish with unique advantages for studying the BRB. The subcellular location of choline transporter-like 1 enhanced green fluorescent protein (CTL1-EGFP) and mCherry-clathrin light chain 2 (CLC2) in a … Recapitulation of fast skeletal muscle development in zebrafish by transgenic expression of GFP under the mylz2 promoter. A: A schematic map of the DNA fragment used in Tol2‐based dat transgenesis. The EGFP coding sequence was cloned in frame into dat exon 1 using an E. coli strain (DY380) with temperature inducible RecET homologous recombination system according to procedures described by Liu et al. Zebrafish models for functional and toxicological screening of nanoscale drug delivery systems: promoting preclinical applications. Adult zebrafish (Danio rerio) were kept at 28°C in a 13-hour light/11-hour dark cycle, and procedures were approved by the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC). Zebrafish is also amenable to genetics due to its relatively short generation time (2–3 months). Transgenic Zebrafish. INTRODUCTIONThe green fluorescent protein (gfp) gene, originally isolated from the jellyfish Aequorea victoria, is widely used as a reporter gene for investigation of tissue-specific gene expression and cellular localization of proteins because the fluorescence of its protein product, GFP, can be conveniently detected in living cells (Prasher et al., 1992;Chalfie et al., 1994;Tsien, 1998). This included the DA neurons in the vDC (groups 2–6). EGFP expression was detected in the ventral-nasal eye at 3 days postfertilization and spread throughout the eye. Double immunostaining for green fluorescent protein (GFP) and tyrosine hydroxylase (TH) on horizontal cryosections of Tg(dat:EGFP) larvae at 5 days post‐fertilization (dpf). Numbers (1–6) indicate different groups of DA neurons in the ventral diencephalon. When the three hybrid GFP constructs were introduced into zebrafish embryos by microinjection, the three promoters were activated faithfully in developing zebrafish embryos. Zebrafish neutrophils (heterophils) are identifiable from approximately 48 hours after fertilization, 2 and the innate immune system exists in isolation from any adaptive system, which does not arise until approximately 4 weeks after fertilization. GFP‐positive cells and TH‐positive cells are shown in green and red respectively. Double fluorescent in situ hybridization was performed as described in MacDonald et al. P values of < 0.05 (two‐sided) were considered as statistically significant. GFP‐positive individuals were raised to sexual maturity and outcrossed with wild type adult fish to identify transgenic carriers. A–D: Sagittal sections of an adult zebrafish hybridized with the krt8 antisense riboprobe. Paraformaldehyde fixation of zebrafish induces strong autofluorescence in the green, red and far red channels. The 3 dpf Tg(dat:EGFP) embryos were transversely cryosectioned and stained with anti‐GFP and anti‐TH antibodies. The GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor on… Dev. This work was supported by a grant from the Canadian Institutes of Health Research to M.E. Delayed effects of methylmercury on the mitochondria of dopaminergic neurons and developmental toxicity in zebrafish larvae (Danio rerio). / Mattingly, Carolyn J.; McLachlan, John A.; Toscano, William A. A,C,E,G,I,K: Lateral views with anterior to the left. Color images are available online. A PAC clone (BUSMP706K0187Q9) was identified to contain the dat gene. Signals were visualized on a Nikon Eclipse E3600 stereomicroscope with filters for both fluorescent stains. This was also confirmed by confocal microscopy analysis of whole‐mount larvae immunostained for both GFP and TH at 3 dpf (Fig. Please check your email for instructions on resetting your password. USA.gov. Functional prediction and physiological characterization of a novel short trans-membrane protein 1 as a subunit of mitochondrial respiratory complexes. In this study, we took advantage of the fact that DAT expression distinguishes DA neurons from other catecholaminergic (CA) neurons in the developing embryo (Holzschuh et al., 2001) and produced a line of transgenic fish in which the green fluorescent protein (GFP) is expressed under the control of regulatory elements of the zebrafish dat gene. This allows us to view the migration of the axons in … 171: 123–129. Y.X. In the central nervous system (CNS), the neurotransmitter dopamine plays important roles in a variety of physiological and behavioral processes such as voluntary movement, cognition, memory and reward (Bjorklund and Dunnett, 2007). The Aequorea victoria green fluorescent protein can be used as a reporter in live zebrafish embryos. 109, No. Green Fluorescent Protein (GFP) is a common protein used to localise proteins, observe protein interactions and quantify gene expression. Jontes JD, Emond MR. Research output: Contribution to journal › Article › peer-review Injected embryos were screened for GFP expression in the desired area between 48 and 72 hpf. Please enable it to take advantage of the complete set of features! Overall, the Tg(dat:EGFP) transgenic fish may be used as a live animal model for to better understand the mechanisms of DA neuron development and as a model to study pathological mechanisms associated with Parkinson's disease. Furthermore, MPTP is metabolized into the toxic MPP+ which, in turn, is taken into DA neurons through Dat. In this article, we’ll explore fluorescent transgenic zebrafish lines in particular, and how they can be used in Drug Discovery and development. 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