ENU-Induced Zebrafish Telomerase Mutant

Zebrafish were maintained in accordance with Institutional and National animal care protocols. The telomerase mutant line tert ^AB/hu3430 possesses a T→A point-mutation in the tert gene. Zebrafish mutant lines were generated by N-Ethyl-N-nitrosourea (ENU) mutagenesis (Utrecht University, Netherlands) [49] (link). Briefly, adult male zebrafish were randomly mutagenized with ENU and outcrossed against wild-type females. A library of F1 animals was then constructed. Genomic DNA of these F1 animals was isolated and arrayed in PCR plates. The DNA was screened for mutations in target genes by re-sequencing or TILLING. Animals with interesting mutations were recovered from the library (either re-identified from a pool of living F1 fish or recovered by in vitro fertilization with frozen sperm) and outcrossed against wild-type fish.
tert ^AB/hu3430 line is available at the ZFIN repository (ZFIN ID: ZDB-GENO-100412-50) from the Zebrafish International Resource Center (ZIRC) and was generously provided to us by Dr. L. Bally-Cuif at the Zebrafish Neurogenetics Department, German Research Center for Environmental Health. The tert^AB/hu3430 used in this paper was subsequently outcrossed 5 times with WT AB for clearing of potential background mutations derived from the random ENU mutagenesis from which this line was originated. The tert^{hu3430/hu3430} homozygous mutant is referred in the paper as tert^−/− and was obtained by incrossing our tert^AB/hu3430 strain. Genotyping was performed by PCR of the tert gene (Table S1) followed by sequencing.
Overall characterization of tert^−/− zebrafish was performed in F1 and F2 animals produced by tert^+/− incross. All 1, 3 and 6 months analysis refers to F1 animals and 12 months analysis refers to F2. The premature death phenotype depicted in Figure 2C refers to F1 animals only