Transgenic Zebrafish Lines for Retinal Studies

Zebrafish were maintained and bred at 26.5°C, and embryos were raised at 28.5°C. The mutant lines used were: nagie oko (nok^m227, a kind gift from Dr Jarema Malicki) and heart and soul (has^m567, a kind gift from Dr Salim Abdelilah-Seyfried). Both represent null alleles of the respective genes. Two transgenic lines were generated in our laboratory: Tg(pBatoh7:gap43-gfp)^cb1 ('ath5:gap-gfp') and Tg(pBatoh7:gap43-rfp)^cb2 ('ath5:gap-rfp'). They express a fluorescent protein (enhanced green fluorescent protein (EGFP) or monomeric red fluorescent protein 1 (mRFP1), respectively) fused to the GAP43 N-terminal palmitoylation signal, under the control of the zebrafish ath5 promoter (comprising 7 kilobases of genomic sequence upstream of the ath5 start codon). For some experiments we used a transgenic line expressing a cytoplasmic form of EGFP under the control of the ath5 promoter ('ath5:gfp', a kind gift from Dr Ichiro Masai) [19 (link)]. The ath5:gap-gfp transgenic line was crossed with carriers of both mutations used, to generate an F₁ generation from which mutant embryos expressing GAP-EGFP in RGCs could be obtained, namely nok^m227 × Tg(pBatoh7:gap43-gfp)^cb1 and has^m567 × Tg(pBatoh7:gap43-gfp)^cb1.