4.1. Zebrafish Husbandry and Manipulation

Wild-type and casper [31] zebrafish were maintained using standard husbandry practices [40]. This included feeding thrice daily with a mixture of live feed (artemia and rotifers) and a dry granulated foodstuff (Otohime Hirame Japan). Embryos were injected with 100 pg/nL mRNA encoding transcription activator-like effector nucleases (TALENs) [41] targeting an NdeI site exon 3 of il2rga, as described [16] and raised to adulthood. Progeny were screened for the presence of mutations by PCR of genomic DNA derived from fin-clips with primers flanking the target site (5′-CGAAGACTGTCCTGAATATGAGAC; 5′-TCTGGTCAGTCCTGTAACGAAC) followed by high resolution melting (HRM) analysis using Precision Melt Suremix and Analysis Software (BioRad, Hercules, CA, USA) [42] and confirmed by restriction fragment length polymorphism analysis with NdeI as well as Sanger sequencing (Australian Genome Research Facility, Melbourne, Australia). Confirmed mutants were out-crossed with wild-type fish for two generations with heterozygote progeny ultimately in-crossed to generate homozygous mutants or crossed onto the casper background.