Maintaining stocks and experimental lines of telomerase mutants. (A) Breeding stocks of tert^+/− zebrafish must be generated by outcrosses to wild-type (WT; tert^+/+) fish. WT zebrafish used for this should be from a stock with verified telomere length. The tert^+/− progeny can be used in consecutive crosses for stock maintenance, as outbreeding with the WT stocks introduces new ‘pools’ of telomeres with diverse length. This minimises (but does not completely eliminate) telomere shortening across generations. All generations of tert^+/− zebrafish from stock maintenance can be used to produce experimental lines, but the age of the parents used must be consistent. (B) Crossing tert^+/− zebrafish generated from stock maintenance (G0) will produce tert^+/+, tert^+/−and tert^−/− progeny (G1). Owing to haploinsufficiency, G0 tert^+/− zebrafish will have reduced capacity to extend telomeres, so G1 tert^+/− as well as tert^+/+ zebrafish will inherit shorter telomeres (indicated by dotted lines), with G1 tert^+/− zygotes having reduced capacity to extend their telomeres. Therefore, tert^+/− zebrafish incrosses must not be used for stock maintenance. As tert^−/− zebrafish completely lack telomerase, they will have even shorter telomeres and associated phenotypes will be apparent in the first generation. G0, generation 0; G1, generation 1; tert, telomerase reverse transcriptase; WT, wild type.