Function And Regulation Of MicroRNAs In Embryonic Hematopoeisis

microRNAs are short single-strand non-coding ribonucleic acid (RNA) molecules, on average about23nucleotides long and are found in eukaryotic cells and are mainly transcribed by RNA polymerase Ⅱ (Pol Ⅱ), then digested by RNAase Ⅲ (Drosha and Dicer). Mature microRNA’s target sites are in the three prime untranslated regions (3’UTR) of the mRNA, and usually resulting in translational repression and gene silencing. microRNA participate in many biological progress, such as organ formation and tissue differentiation. According to paper reports, at least30microRNAs have special expression profiles in adult human bolld cells, and many of them are involved in the differentiation of hematopoietic stem cells as well as interacting with key factors in embryonic hematopoiesis. However, their roles in embryonic hematopoiesis still needed further research.In order to reveal microRNA’s function in embryonic hematopoiesis, we used embryonic body which were induced to differentiate from mouse embryonic stem cells as the model of embryo development. In the first step, we picked candidate microRNAs that had special expression profiles in embryonic hematopoiesis based on previous data. In the second step, in order to confirm whether these candidate microRNAs were able to modulate embryonic hematopoiesis, we uesd retro virus vector to over-express candidate microRNA in mouse embryonic stem cells. Then we succeed to construct inducible mouse embryonic stem cell line which were able to over-express candidate microRNAs under doxycyline induction. These cell lines were induced to differentiated into embryonic body and induced to over-express candidate microRNAs in certain times during differentiation to analyze hematopoietic factors by QPCR and FACS. From these data, we got possible targets of candidate microRNAs, and our next step is to confirm it and find out their direct targets.Also, in order to study microRNAs in human embryonic stem cell, we constructed a homologus recombination construct which contained tet-on promoter, Hygromycine resistance gene, loxP and Frt, etc to target3’UTR of human Actb gene. After homologus recombination, the gene of interest or microRNA were able to be inserted in the targeted site and over-expressed under doxycyline induction. It was an useful tool to study the function of genes and microRNAs in human embryonic stem cells.