RESUMO
The widespread availability of programmable site-specific nucleases now enables targeted gene disruption in the zebrafish. In this study, we applied site-specific nucleases to generate zebrafish lines bearing individual mutations in more than 20 genes. We found that mutations in only a small proportion of genes caused defects in embryogenesis. Moreover, mutants for ten different genes failed to recapitulate published Morpholino-induced phenotypes (morphants). The absence of phenotypes in mutant embryos was not likely due to maternal effects or failure to eliminate gene function. Consistently, a comparison of published morphant defects with the Sanger Zebrafish Mutation Project revealed that approximately 80% of morphant phenotypes were not observed in mutant embryos, similar to our mutant collection. Based on these results, we suggest that mutant phenotypes become the standard metric to define gene function in zebrafish, after which Morpholinos that recapitulate respective phenotypes could be reliably applied for ancillary analyses.
Assuntos
Desoxirribonucleases/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes/métodos , Morfolinos/farmacologia , Mutação/genética , Oligonucleotídeos Antissenso/farmacologia , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Western Blotting , Desoxirribonucleases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fenótipo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/antagonistas & inibidoresRESUMO
Atherogenesis is a chronic inflammatory response and intercellular adhesion molecule (ICAM-1) induced by cytokines plays a role in this event. In this study, the molecular mechanisms of tumor neurosis factor alpha (TNFalpha)- and IL-6-induced ICAM-1 gene expression in endothelial cells (ECs) were examined. ECs infected with adenovirus carrying the dominant negative mutant of Rac (Ad-RacN17) exhibited inhibition in both TNFalpha- and IL-6-induced ICAM-1 expression. Consistently, ECs transfected with RacN17 inhibited both TNFalpha- and IL-6-induced ICAM-1 promoter activities. Functional analysis of ICAM-1 promoter, however, indicated that the cis-acting elements in response to TNFalpha and IL-6 are different. The NFkappaB binding site in the ICAM-1 promoter region was crucial for TNFalpha-induced ICAM-1 expression but not for the induction by IL-6. ECs infected with Ad-RacN17 attenuated the TNFalpha-induced NFkappaB binding activity. In contrast, IL-6 activated a transcriptional factor, signal transducer and activator of transcription-3 (Stat3) via the phosphorylation of Tyr705 at Stat3. ECs transfected with the dominant negative mutant of Stat3 (Stat3F) demonstrated that Stat3 was required for IL-6-induced ICAM-1 gene expression. Interestingly, the phosphorylation of Tyr705 and Ser727 in Stat3 was greatly inhibited in IL-6-treated ECs previously infected with Ad-RacN17. Our data strongly indicated that ICAM-1 gene induction by TNFalpha and IL-6 is mediated mainly via NFkappaB and Stat3, respectively and Rac1 appears to play a central role in modulating cytokine-induced ICAM-1 expression in ECs.