To create mutant alleles in zebrafish, we injected the CRISPR/sgRNA stock volume with concentrations given in Table 2. Chapter 2 - Breeding Detailed Methods for Breeding Over Marbles (Source: S. Russell) Fish and marbles At the onset of the light cycle, zebrafish will generally initiate breeding behavior that results in the laying and fertilization of eggs. Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Further examinations by a PCR-based “blind assay" and array comparative genomic hybridization both failed to find universal sex-linked differences between the male and female genomes. 5 Setup of the tanks used for breeding zebrafish in the Berlin labora-tory (courtesy of Dr. T. Meinelt, IGB) 29 Fig. Zebrafish (Danio rerio) is a fresh water fish that inhabits rivers in India, Pakistan and other places in Asia. Freshly laid fertilized eggs are collected from breeding tanks and injected with the CRISPR/sgRNA stock volume into the blastomeres using a microinjection setup. Sperm can be extracted from an anesthetized male zebrafish by drying its urogenital pore before covering it with the open end of a capillary tube or pipette tip ( Fig. Indeed, they have optimized CRISPR genome editing protocols for zebrafish that allow disruption of gene function at nearly 100% efficiency and even demonstrate deletion of genomic DNA more than 50 kb in length. However, little is known on the nutritional requirements of zebrafish [29, 30], being mostly reared with information available for Cyprinaformes [30, 31]. Consequently, Zebrafish has become one of the ideal organisms to consider in studying the biological processes. Variation among family sex ratios was reduced after selection for breeding pairs with predominantly male or female offspring, another indication that zebrafish sex is regulated genetically. This volume collects sophisticated and practical techniques, including several not yet widely known, from laboratories around the world. Protocol revised: 6/26/2007 Protocol written by: Natasha Meyer In the afternoon (anytime after feeding) of the day before you want to breed, pair 1 male and 2 females in a breeding chamber (these hook into the larger tanks). Fill each tank with water from the aquaculture system. In Zebrafish: Methods and Protocols, expert researchers provide a collection of experimental techniques for the genetic modification, manipulation, and in vivo live imaging of zebrafish. Principal investigators (PIs) ... Zebrafish: Zebrafish are external fertilizers. Using a fish net, transfer one adult male and one to two adult females to inner container in each breeding tank. It only takes 36 hours for all of the major organs to develop and 96 hours for them to be properly formed. In the last 20 years, research activity using the zebrafish Danio rerio has increased dramatically, due in part to the ease of breeding and raising them, their genetic tractability, embryonic accessibility, and their imaging potential. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. it became essential to find better protocols and methods to breed and raise zebrafish faster, more reliably and with better welfare. Zebrafish Care Standards . However, a description of their use may be necessary as part of a complete description of experimental protocols involving adult breeding zebrafish. In the last 20 years, research activity using the zebrafish Danio rerio has increased dramatically, due in part to the ease of breeding and raising them, their genetic tractability, embryonic accessibility, and their imaging potential. ability to manipulate the genome. Zebrafish (Danio rerio) are widely available from local pet stores for about a dollar each. Thank you. Simplified protocol for setting up and breeding zebrafish. On the afternoon prior to the day of the chemical screen, set up 10 to 20 zebrafish breeding tanks. Purpose . This is becoming a major I. In "Zebrafish: Methods and Protocols", expert researchers provide a collection of experimental techniques for the genetic modification, manipulation, and in vivo live imaging of zebrafish. This protocol explains how to: breed the zebrafish to obtain embryos. All of the needed equipment, food, and supplies that are necessary for maintaining and breeding them can also be … Most protocols start by setting up fish in an off-system breeding tank with a divider separating males and females. 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