OBJECTIVESTo establish a method of gene analysis via zebrafish model to explore the effect of microRNA-191(miR-191) on myelopoiesis.

METHODSThe 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.

RESULTSThe 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.

CONCLUSIONThe 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.