Expression and Characterization of Bioactive Human Thrombopoietin in the Milk of Transgenic Mice

Abstract

Human thrombopoietin (hTPO) is the primary physiological regulator of platelet production and plays a pivotal role in promoting the proliferation and maturation of megakaryocytic progenitor cells and megakaryocytes. In this study, transgenic mice were produced harboring either full-length or the erythropoietin (EPO)-like amino-terminal domain of hTPO cDNA sequences fused to 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 mouse. The full-length form induced efficient expression of the protein with the highest expression level of 1500 mu g/ml; however, the EPO-like domain alone expressed the protein at <0.1 mu g/ml. The proteins from the two recombinant 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 proliferation of the TPO-dependent BaF3-Mpl cells. A positive correlation appeared between the amount of TPO in the milk of lactating animals and their blood platelet levels. About a twofold increase in platelet numbers in the blood was 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 anticipate that the recombinant hTPO produced efficiently in milk of transgenic mice will have the same activities as the native hTPO in a few in vivo as well as in vitro biochemical aspects.