Chapter 2 36 proximal tubular reabsorption. Thus, it provides a robust and versatile model that can be used for the study of the pathophysiological aspects of cystinosis and for the in vivo screening of novel therapeutic agents. Materials and methods Fish maintenance and breeding The animal care and experimental procedures were carried out in accordance with the ethical committee guidelines for laboratory animal experimentation at KU Leuven and the reference European directive: DIRECTIVE 2010/63/EU on the protection of animals used for scientific purposes. Zebrafish (Danio rerio) used in the current study were AB strain wild-type and ctns‒/‒ mutant initially purchased as heterozygous (ctns‒/+) from the European Zebrafish Resource Centre (EZRC), Karlsruhe, Germany. Cystinotic and wt adult fish were raised at 28.5°C, on a 14/10 hour light/dark cycle under standard aquaculture conditions58. By the 3rd generation in our facility, we could isolate sufficient numbers of mutant homozygous male and female zebrafish and mating was performed only between homozygous adult fish (ctns‒/‒). A mating setting always included four females and two males. After mating, every 50-60 fertilized embryos were transferred into a fresh 10 cm petri dish which was approximately ¾ filled with clean egg water (Instant Ocean Sea Salts, 60 µg/ml) and methylene blue (0.3 ppm). Embryos were sorted out for debris and unfertilized eggs and incubated at 28.5°C. Every day the medium was refreshed, the debris was removed and dead embryos were sorted out. All experiments were performed between 3rd and 6th days post fertilization. Zebrafish genotyping For determining the genotype of adult zebrafish, we extracted DNA from the tail (caudal) fin of live adult fish. DNA was separated by the Wizard SV genomic DNA purification system for animal tissues (Promega, Madison, WI, USA) according to manufacturer’s protocol. Exon 8 PCR of zebrafish ctns gene was performed using: 5′-AGTACAGCGATTACTTAACAGGT-3′ and 5′-GACACCCAGTTTAATGTAGGA-3′ as forward and reverse primers, respectively. PCR products were prepared for sequencing through the Big Dye Terminator technology and sequenced on ABI 3100 sequence analyser (Applied Biosystems, Carlsbad, CA, USA). Data were analysed using SEQUENCE Pilot (JSI Medical Systems, Kippenheim, Germany). Morpholino Fluorescein tagged antisense morpholino oligonucleotides targeting the 5’ UTR of zebrafish ctns mRNA and a control morpholino were obtained from GeneTools
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