Objectives: To establish a method of gene analysis via zebrafish model to explore the effect of microRNA-191(miR-191) on myelopoiesis.
Methods: The hsa-miR-191 or cel-miR-67 was microinjected into the fertilized eggs of zebrafish, then the total RNA of embryos was extracted at 10 s, 24, 36 and 48 hpf for qRT-PCR to detect the expression levels of erythroid and granulocytic genes. Then embryos at the time-point of 24 hpf were collected for whole mount in situ hybridization to detect spatiotemporal expression of those genes.
Results: The antisense RNA probes with high sensitivity and specificity of erythroid genes (gata 1, scl, hbbe 3, lmo 2) and myelomonocytic genes (pu.1, L-plastin, mpx, cebpα) in zebrafish were obtained by molecular cloning and T7 RNA polymerase reaction; the expression levels of the erythroid and myelomonocytic genes of zebrafish microinjected with miR-191 mimics displayed higher than those in control group at the time-points of 24 hpf and 36 hpf. The spatiotemporal expression level of L-plastin at the time-point of 24 hpf was up-regulated, and the other genes were not significantly changed. It was worth mentioning that the mRNA expression level of mpx was significantly up-regulated by 10-20 times at the time-point of 10 s.
Conclusion: The genetic analysis method of embryonic myeloid differentiation has been set up via zebrafish. Preliminary analysis of regulation in zebrafish myelopoiesis shows that miR-191 may be involved in the regulation of erythroid and myelomonocytic differentiation. The mechanism and corresponding function of mpx regulated by the other factors need to be further studied.