Example 18
Lines were raised and maintained following standard literature practice and in accordance with the Guide for the Care and Use of Laboratory Animals provided by the University of Southern California. Fish samples were part of a protocol approved by the IACUC (permit number: 12007 USC).
Transgenic FlipTrap Gt(desm-Citrine) ct122a/+ line is the result of previously reported screen, Tg(kdrl:eGFP)s843 line was provided by the Stainier lab (Max Planck Institute for Heart and Lung Research). The Tg(ubi:Zebrabow) line was a kind gift from Alex Schier. Controllable recombination of fluorophores was obtained by crossing homozygous Tg(ubi:Zebrabow) adults with a Tg(hsp70I:Cerulean-P2A-CreERT2) line. Embryos were raised in Egg Water (60 μg/ml of Instant Ocean and 75 μg/ml of CaSO4 in Milli-Q water) at 28.5° C. with addition of 0.003% (w/v) 1-phenyl-2-thiourea (PTU) around 18 hpf to reduce pigment formation.
Zebrafish samples with triple fluorescence were obtained by crossing Gt(desm-Citrine)ct122a/+ with Tg(kdrl:eGFP) fish followed by injection of 100 μg per embryo of mRNA encoding H2B-Cerulean at one cell stage as described in previous work29. Samples of Gt(desm-Citrine)ct122a/+;Tg(kdrl:eGFP); H2B-Cerulean were imaged with 458 nm laser to excite Cerulean, Citrine and eGFP and narrow 458-561 nm dichroic for separating excitation and fluorescence emission.
