Cflarb complemented the function of cflara to allow cflara knock out zebrafish to normal development

Abstract

Cellular FLICE-inhibitory protein (cFLIP, cflara) is a regulator of death receptor (DR)-induced apoptosis and NF-κBactivation. cFLIP is known to prevent activation of the caspase cascade by binding to FADD/caspase-8. Up-regulatedcFLIP has been identified in many tumor types, and therefore restoring apoptosis by silencing cFLIP may be one of themore potent strategies in cancer therapeutics. The zebrafish cFLIP gene, cflara, has 2 death effector domains (DEDs)and a single caspase-like domain. Expression of cflara was detected in the zebrafish embryo by RT-PCR and wholemountin situ hybridization. To study the in vivo function of cflara, we generated a cflara knockout mutant zebrafishusing transcription activator-like effector nucleases (TALENs). Frame shift mutation is caused by a 10 bp deletion in thefirst DED domain. By inbreeding the F1 generation, a homozygous mutant fish was produced and confirmed by PCR.Knockout of cflara leads to abnormal heart development and embryonic lethality in mice. However, mutant zebrafishdid not show any differences from wild type in heartbeat rate, survival rate or development. Zebrafish have analoguesof both cflara and cflarb. Quantitative PCR showed that cflarb mRNA levels of mutant zebrafish were higher than thosein the wild type. In a chemical exposure experiment, mutant zebrafish larvae showed a longer survival rate comparedwith wild type after CoCl2 treatment. However, no significant difference was observed from cisplatin treatment. Thisdata suggests that cflarb may contribute to normal development and causes a difference in chemical resistance.