Résumé
Objective To establish the cell models of c-myb+/+ and c-myb+/-ES in vitro by gene targeting with ES cell culture system, with the aim to examine the detailed role of the transcription factor c-myb in haemopoietic commitment and differentiation. Methods Haemopoietic differentiation was initiated by seeding ES cells in LIF-free methylcellulose medium. The embryoids of c-myb+/+ and c-myb+/-ES were analyzed by methylcellulose colony assay and real-time PCR, the formative process of embryoids and the expression of relative genes were compared in each haemopoietic system at different differentiation stages and procedures. Results The formative process of embryoids was similar for both c-myb+/+ and c-myb+/-ES cells, but the size and frequency of EBs were reduced in the case of the c-myb+/-cells. Similar kinetics gain existed for the formation of CFU-Es in both groups, but the number in c-myb+/-group was less than that of c-myb+/+ group, and the colonies were generally smaller. BFU-E was first detectable on day 7, and the peak value emerged on day 10 in both groups. Similar kinetics gain existed for the formation of CFU-M in the two groups, but the number was larger in c-myb+/-group than that in c-myb+/+ group, while the number of CFU-GM in c-myb+/-group was less than that in c-myb+/+ group. Real-time PCR analysis showed no changes on the gene expressions of ?-globin, ?-globin, GATA3, CD34, Lys, c-fms and c-mpl in both groups. Conclusions Sub-level expressions of c-myb (55%) and c-myb+/-are sufficient to allow progenitor to expand, but they throw a negative influence on the terminal differentiation. Sub-level expression of c-myb may influence the erythropoiesis and granulocytic development, but throw no influence on the precursor cells to differentiate into macrophages. The expressive levels of c-myb have no effect on the expressions of ?-globin, ?-globin, GATA3, CD34, Lys, c-fms and c-mp.
Résumé
Objective The c-myb is an important transcription factor in early haemopoietic system development and differentiation. We wish to use ES cell culture system by gene target getting cell models of c-myb+/+ and c-myb-/- ES in vitro in order to examine the detailed roles of the transcription factor c-myb in haemopoietic commitment and differentiation. Methods Haemopoietic differentiation was initiated by seeding ES cells in methylcellulose medium in the absence of LIF. The embryoid bodies of c-myb+/+ and c-myb-/- ES were analyzed by methylcellulose colony assay and real-time PCR to compare the formation of each haemopoietic system on different differentiation stage and procedure and relative gene expression. Results The formation of embryoid bodies was similar for both c-myb+/+ and c-myb-/- ES cells with the exception that the size and frequency of EBs reduced in the case of the c-myb-/- cells. The number of embryoid bodies increased more markedly in c-myb+/+ than that in c-myb-/-. Erythroid progenitors (CFU-E) were first present in c-myb+/+ group at 6 d after in vitro differentiation, and their number reached the peak at 8 d. Similar kinetics were seen for the formation of CFU-Es in c-myb-/- group, but their number was lower than that in c-myb+/+ group, and the colonies were generally small. CFU-M were first detectable at 7 d, and the peak levels were present at 7 d in c-myb+/+ group. Similar kinetics were seen for the formation of CFU-M in c-myb-/- group, but their number was lower than that in the c-myb+/+ group. CFU-GM was first detectable at 7 d, and the peak levels were present at 12 d in c-myb+/+ group. CFU-GM was not detected in c-myb-/- group. Real-time PCR analysis showed that there was no change in the gene expressions of ?-globin, zeta-globin, Lys, and C-fms in the two groups of c-myb+/+ and c-myb -/-. Conclusion Low levels of c-myb are suff-icient to allow progenitor expansion, but progression of progeniters towards terminal differentiation is significantly altered. Low levels of c-myb can influence erythropoiesis development, but precursor cells capable of differentiating into macrophages are present in haemopoietic commitment. The levels of c-myb can not change the gene expressions of ?-globin, zeta-globin, Lys, and C-fms.