Development of BMP7-producing human cells, using a third generation lentiviral gene delivery system.
Bone morphogenetic protein 7 (BMP7), a member of the transforming growth factor β (TGF-β) superfamily, plays important roles in the development of various tissues and organs in mouse and human. In particular, BMP7 is critical for the formation of the nervous system and it is considered to have therapeutic potential in brain injury and stroke. One approach to make BMP7 more suitable for therapeutic purposes is the development of efficient vectors that allow the consistent, reliable and cost-effective production of the BMP7 protein. In this study, we developed an efficient BMP7 delivery system, using a third generation lentiviral vector to produce functional BMP7 protein. The lentiviral transduction of several human cell types, including human embryonic kidney 293 (HEK293) cells, amniotic fluid cells, NTera2 neurons (NT2-N) and primary neuronal cultures resulted in BMP7 expression. The production of BMP7 protein was achieved for at least 4 weeks post-transduction, as determined by enzyme-linked immunosorbent assay (ELISA). SMAD phosphorylation and neuronal differentiation assays verified the bioactivity and functionality of the lentiviral-based BMP7 protein, respectively. In addition, the intracerebroventricular injection of the lentivirus resulted in exogenous BMP7 expression in both neurons and astrocytes in the mouse brain. Taken together, this gene delivery system provides a reliable source of functional BMP7 protein for future in vitro and in vivo studies.
Neurogenesis and Brain Repair Group, Neurobiology Program, Institute for Biological Sciences, National Research Council Canada, Ottawa, ON K1A 0R6, Canada.
David W
DW
Chitty
Roger G
RG
Tremblay
Maria
M
Ribecco-Lutkiewicz
Julie
J
Haukenfrers
Bogdan
B
Zurakowski
Bernard
B
Massie
Marianna
M
Sikorska
Mahmud
M
Bani-Yaghoub
D000653:Amniotic Fluid / Q000166:cytology; D000818:Animals; D015153:Blotting, Western; D055419:Bone Morphogenetic Protein 7 / Q000096:biosynthesis*; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000008:administration & dosage / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D006801:Humans; D007150:Immunohistochemistry; D007276:Injections, Intraventricular; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D009474:Neurons / Q000378:metabolism / Q000502:physiology; D011247:Pregnancy; D051785:Smad Proteins / Q000096:biosynthesis / Q000235:genetics; D014161:Transduction, Genetic; D014162:Transfection / Q000379:methods*
D055419:Bone Morphogenetic Protein 7; D018076:DNA, Complementary; D051785:Smad Proteins; D049452:Green Fluorescent Proteins