Analysis of placental regulation of hematopoiesis.
Placental hormones contribute to changes in maternal physiology, especially to changes in the blood system. Methods are described to express a placental hormone from a cloned cDNA by transfection into a mammalian cell line, to purify the hormone, and to assess the activities of the hormone in primary mouse bone marrow cell cultures. The example used in this chapter is prolactin-like protein F (PLP-F), a recently discovered mouse placental hormone that acts on the myeloid lineage. This hormone has been expressed at high levels in stably transfected Chinese hamster ovary cells. The protein is secreted from these cells after cleavage of the signal sequence and the addition of N-linked carbohydrate. A series of chromatographic steps are used to purify the protein to homogeneity, which is verified by gel electrophoresis and silver staining; the identity of the purified protein is confirmed by immunoblot analysis. Purified protein is then assayed by addition to primary bone marrow cells and scoring the growth and the differentiation of the megakaryocyte progenitor, colony forming unit-megakaryocyte.
Zhou
Beiyan
B
Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL, USA.
Linzer
Daniel I H
DI
D000818:Animals; D016466:CHO Cells; D002845:Chromatography / Q000379:methods; D003114:Colony-Forming Units Assay / Q000379:methods; D006224:Cricetinae; D005260:Female; D005822:Genetic Vectors; D006410:Hematopoiesis / Q000502:physiology*; D008533:Megakaryocytes / Q000187:drug effects; D051379:Mice; D010920:Placenta / Q000378:metabolism; D010926:Placental Hormones / Q000096:biosynthesis* / Q000302:isolation & purification / Q000502:physiology; D010957:Plasmids; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D014162:Transfection / Q000379:methods
D010926:Placental Hormones; D011257:Pregnancy Proteins; C502151:Prlpf protein, mouse; D011994:Recombinant Proteins