Zebrafish, a model of choice for biomedical research. Yoav Gothilf Dept. Neurobiology, Tel Aviv University
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1 Zebrafish, a model of choice for biomedical research Yoav Gothilf Dept. Neurobiology, Tel Aviv University yoavgothilf@gmail.com
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7 Zebrafish Vertebrate External fertilization Many embryos Transparent embryos Fast development Short generation time Easy to maintain
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14 Zebrafish, a model of Choice Vertebrate Large number of accessible transparent embryos Fast development and short generation time Developmental mutants Gene expression analysis Transgenesis Gene knockdown, knockout and knockin Behavioral tests Imaging capabilities
15 Whole genome duplication is a rare evolutionary event that has played a dramatic role in diversification by providing material for evolution of new gene functions. A duplicated gene provides a greater, less-constrained chance for natural selection to shape a novel function
16 Whole mount in situ hybridization Substrate AP AP Precipitate DIG DIG Probe mrna
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19 Digoxigenin (DIG) is a steroid found exclusively in the flowers and leaves of the plants Digitalis purpurea, Digitalis orientalis and Digitalis lanata (foxgloves), where it is attached to sugars
20 GnRH3 mrna localization during zebrafish development 1 dpf 3 dpf 5 dpf T 15 dpf 23 dpf 5dpf 25 dpf T T T POA Mes Ori Palevitch 2005
21 AANAT2 mrna expression in zebrafish larvae ZT6 ZT18 LD DD Gothilf et al., 1999
22 Transgenesis: Method development and utilization for visualizing and manipulating neuros
23 promoter EGFP
24 Transgenic fish in which GFP is driven by vascular-specific promoters
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28 Kawakami 2005 Discovering the Tol2 transposase system
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30 Gene and enhancer trap using the Tol2 system Kawakami 2005
31 The GAL4-UAS system
32 Gene and enhancer trap using the Tol2 system Gal4 Gal4 Gal4 Kawakami 2005
33 Specific neuronal inhibition
34 Neuronal ablation Metronidazole Nitroreducdase (nfsb gene) Cell death
35 Synaptic plasticity
36 Lior Appelbaum, BIU
37 Shainer et al., 2017
38 Blocking the clock DCLK
39 Seeing neurons in action, using Ca ++ sensor
40 Activating neurons ChR
41 Optogenetics Light sensitive single-cell algae Light sensitive sodium channel called channelrhodopsin
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43 Generation of action potential by light pulse Channelrhodopsin Halorhodopsin The blue-light sensitive Channelrhodopsin and the yellow light-activated chloride pump halorhodopsin together enable activation and silencing of neural activity
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47 Gene knockdown for studying gene function Using morpholino-modified oligonucleotides (MO)
48 Gene knockdown for studying gene function Using morpholino-modified oligonucleotides (MO) Fw Rev Introne 1 Esone 1 Esone 2 SPL1 Fw Rev Introne 1 Esone 1 Esone 2 SPL2
49 Muscle-specific expression of Smyd1 21h 24h 24h heart
50 Morpholino (MO)-modified oligonucleotides
51 Knockdown of Smyd1 expression disrupts muscle cell differentiation MO A B C D Control
52 Knock down of Smyd1: Larvae are paralyzed and heart doesn t beat
53 GNE-mediated Hereditary Inclusion Body Myopathy GNE (UDP N-Acetylglucosamine 2-epimerase/N-Acetylmannosamine kinase) Daya et al., 2014
54 GNE MO knockdown phenotypes 1mM Splice Morpholino - 3dpf Control Mild Intermediate Severe
55 Larvae locomotor activity Immunostaining of 7 dpf larvae with slow muscle myosin antibody Daya et al., 2014
56 Monitoring locomotor activity of zebrafish larvae Danio Vision, Noldus
57 Vision test
58 IQ test for fish A. 20cm 20cm 11cm v 20cm 30cm
59 Latency to red zone (sec) IQ test for fish WT Sib Trail
60 Gene knockout for studying gene function CRISPR Cas9 TALEN
61 clustered regularly interspaced short palindromic repeats (CRISPR)
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63 23 bp
64 Cell size modifications Transcription Activator-Like Effector - TALE
65 TALE DNA-targeting domains contain repeats of 34 aa each. Each repeat displays a different pair of residues at positions 12 & 13 (RVD) that associates preferentially with different nucleotides. H-histidine; D-aspartate; N-asparagine; G-glycine; I-Isoleucine
66 34 aa=102 nt
67 TALEN-mediated Gene Knockout Gene of interest TALEN Left DNA Binding Domain FokI FokI TALEN Right DNA Binding Domain TALEN domains bind to a gene of interest and FOKI nucleases induce a double-stranded break.
68 TALEN-mediated Gene Knockout Left DNA Binding Domain FokI FokI Right DNA Binding Domain TALEN domains bind to a gene of interest and FOKI nucleases induce a double-stranded break.
69 TALEN-mediated Gene Knockout Nonhomologous end joining Nonhomologous end joining Non-homologous end joining (NHEJ) repair system introduces indels (insertions and/or deletion) into the gene sequence.
70 TALEN-mediated Gene Knockout Mutated (inherited) gene sequence Non-homologous end joining (NHEJ) repair system introduces indels (insertions and/or deletion) into the gene sequence.
71 Construction of TALE arrays Left TALE array Right TALE array Ligation step 1 Ligation step 2 Ligation step 3 Ligation step 4
72 What are the advantages and disadvantages of zebrafish as a model for human genetic diseases? Whole genome duplication: What could be the consequence and why is this a consideration? What are the advantage of CRISPR over TALEN? What are the advantage of TALEN over CRISPR? Which of the two systems is mostly used in zebrafish research?
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