RESUMO
Most current methods for genotyping zebrafish embryos require sacrifice or raising the animal to at least 1 month of age for fin amputation. These limitations restrict the use of zebrafish and increase time and costs for experiments. This article introduces an innovative method for genotyping live zebrafish embryos. The method utilizes enzyme to extract a small amount of genetic material from the skin tissue of the embryo. Then, using conventional polymerase chain reaction (PCR) strategy, the embryo is genotyped. This approach was successful >95% of the time while maintaining high viability (>90%) of the embryo. This effective method can facilitate high-throughput screening and other applications in zebrafish.
Assuntos
Genótipo , Técnicas de Genotipagem/veterinária , Peixe-Zebra/genética , Animais , Embrião não Mamífero/enzimologia , Técnicas de Genotipagem/métodos , Reação em Cadeia da Polimerase/métodosRESUMO
Ventricular trabeculation is one essential step for generating a functionally competent ventricular wall, while how the early trabeculae carneae forms and subsequently develops into mature chambers is poorly understood. We found that in zebrafish zfpm1-/- juvenile, cardiac function is significantly compromised, with hearts exhibiting deformed trabecular meshwork. To elucidate the mechanisms of Zfpm1 function in cardiac trabeculation, we analyzed zfpm1 mutant hearts more closely and found that loss of Zfpm1 activity resulted in over-activation of Neuregulin-ErbB signalling and abnormally elevated cardiomyocyte proliferation during cardiac trabeculae growth and modeling stages. These results implicate Zfpm1 plays a pivotal role in coordinating trabeculae patterning and growth.
Assuntos
Miocárdio/metabolismo , Neurregulinas/genética , Receptor ErbB-2/genética , Transdução de Sinais/genética , Proteínas de Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Proliferação de Células/genética , Células Cultivadas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Coração/embriologia , Microscopia Confocal , Miocárdio/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Neurregulinas/metabolismo , Receptor ErbB-2/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Production of all-male and sterile fish may not only substantially improve yield but also be crucial for the application of genome modified species in aquaculture. Previously, it was reported that the fish lacking primordial germ cells (PGCs) becomes infertile, and nitroreductase, an enzyme converting non-toxic metronidazole (MTZ) into toxic metabolites, induces targeted toxicity to kill the cells expressing it. In this study, we generated a transgenic zebrafish line of Tg(nanos3:nfsB-mCherry-nanos3 3'UTR) in which the NfsB nitroreductase is solely expressed in PGCs. Treating the embryos derived from the female transgenic zebrafish with MTZ from 0 through 2 dpf (days post fertilization), we found that the germ cells were completely eliminated in the ones older than 2.5 dpf. At 20 dpf, the MTZ-treated juvenile had no germ cells in their gonads. At 100 dpf, the MTZ-treated adult exhibited male-like morphology and showed normal mating behaviors although they had no germ cells but only supporting cells in their gonads. Taken together, our results demonstrated that conditional elimination of PGCs during early development make the zebrafish male-like and infertile. It may provide an alternative strategy to make sterile and all-male farmed fish that is good for increasing aquaculture yield and preventing the genome modified species from potential ecological risks.
