OBJECTIVE: The primary objective of this study was to identify and clone the first nonmammalian thrombopoietin (TPO), chicken TPO, and its receptor c-Mpl for the purpose of characterizing their activities both in vitro and in vivo. MATERIALS AND METHODS: Chicken TPO was cloned using the methods of reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends. Northern blotting and RNAse protection assays were employed to analyze the levels of RNA expression in a panel of tissues and cell lines. To study cell surface expression of c-Mpl, polyclonal antibodies were prepared against bacterially derived c-Mpl. Both baculovirus-derived recombinant TPO and retrovirally expressed TPO and c-Mpl were used for the in vivo experiments. RESULTS: Both chicken TPO and its receptor c-Mpl were identified and cloned. Expression of chicken TPO was restricted to only the liver and spleen, while c-mpl was expressed in the bone marrow, lung, and spleen. In vitro experiments with sorted multipotent chicken bone marrow-derived progenitors demonstrated that TPO plays a role in the commitment of these cells to the thrombocytic lineage. Furthermore, TPO in cooperation with stem cell factor also supports proliferation of multipotent progenitors. In experimental animals, the intravenous application of recombinant chicken TPO or overexpression of TPO and c-mpl via retroviral infection lead to erythroblastosis and thromboblastosis. CONCLUSION: The characterized chicken thrombopoietin and its receptor c-Mpl will be valuable tools to further study thrombocytic differentiation and hematopoietic stem cell development. Moreover, the introduced experimental model of the chicken bipotent thrombo-/erythropoietic-progenitor can be used to identify key regulators of cell fate determination.
OBJECTIVE: The primary objective of this study was to identify and clone the first nonmammalian thrombopoietin (TPO), chickenTPO, and its receptor c-Mpl for the purpose of characterizing their activities both in vitro and in vivo. MATERIALS AND METHODS:ChickenTPO was cloned using the methods of reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends. Northern blotting and RNAse protection assays were employed to analyze the levels of RNA expression in a panel of tissues and cell lines. To study cell surface expression of c-Mpl, polyclonal antibodies were prepared against bacterially derived c-Mpl. Both baculovirus-derived recombinant TPO and retrovirally expressed TPO and c-Mpl were used for the in vivo experiments. RESULTS: Both chickenTPO and its receptor c-Mpl were identified and cloned. Expression of chickenTPO was restricted to only the liver and spleen, while c-mpl was expressed in the bone marrow, lung, and spleen. In vitro experiments with sorted multipotent chicken bone marrow-derived progenitors demonstrated that TPO plays a role in the commitment of these cells to the thrombocytic lineage. Furthermore, TPO in cooperation with stem cell factor also supports proliferation of multipotent progenitors. In experimental animals, the intravenous application of recombinant chickenTPO or overexpression of TPO and c-mpl via retroviral infection lead to erythroblastosis and thromboblastosis. CONCLUSION: The characterized chickenthrombopoietin and its receptor c-Mpl will be valuable tools to further study thrombocytic differentiation and hematopoietic stem cell development. Moreover, the introduced experimental model of the chicken bipotent thrombo-/erythropoietic-progenitor can be used to identify key regulators of cell fate determination.
Authors: Ondřej Svoboda; David L Stachura; Olga Machoňová; Petr Pajer; Jiří Brynda; Leonard I Zon; David Traver; Petr Bartůněk Journal: Blood Date: 2014-05-28 Impact factor: 22.113