Expression and characterization of bioactive human thrombopoietin in the milk of transgenic mice

Human thrombopoietin (hTPO) is the primary physiological regulator of plate let production and plays a pivotal role in promoting the proliferation and maturation of megakaryocytic progenitor cells and megakaryocytes, In this s tudy, transgenic mice were produced harboring either full-length or the ery thropoietin (EPO)-like amino-terminal domain of hTPO cDNA sequences fused t o the regulatory elements of the bovine beta-casein gene. The transgene RNA was expressed exclusively in the mammary glands of eight transgenic mice, and a trace amount of the transgene was also found in the lungs of one mous e, The full-length form induced efficient expression of the protein with th e highest expression level of 1500 mu g/ml; however, the EPO-like domain al one expressed the protein at <0.1 mu g/ml. The proteins from the two recomb inant cDNAs have apparent molecular weights of about 74 and 17 kDa, due to glycosylation in the case of the full-length cDNA. Cell proliferation assay in vitro indicated that both of the recombinant forms stimulated prolifera tion of the TPO-dependent BaF3-Mpl cells. A positive correlation appeared b etween the amount of TPO in the milk of lactating animals and their blood p latelet levels. About a twofold increase in platelet numbers in the blood w as observed after direct subcutaneous injection of the recombinant hTPO at the level of 30 mu g/kg of body weight. On the basis of these results, we a nticipate that the recombinant hTPO produced efficiently in milk of transge nic mice will have the same activities as the native hTPO in a few in vivo as well as in vitro biochemical aspects.