Development of BMP7-producing human cells, using a third generation lentiviral gene delivery system
Autor: | Bogdan Zurakowski, Marianna Sikorska, Bernard Massie, Maria Ribecco-Lutkiewicz, David W. Chitty, Julie Haukenfrers, Mahmud Bani-Yaghoub, Roger Tremblay |
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Rok vydání: | 2012 |
Předmět: |
DNA
Complementary animal structures Bone Morphogenetic Protein 7 Blotting Western Green Fluorescent Proteins Enzyme-Linked Immunosorbent Assay Smad Proteins SMAD Gene delivery Biology Transfection Cell Line Viral vector Mice Pregnancy Transduction Genetic In vivo Animals Humans Human amniotic fluid cells Signalling pathway Cells Cultured Injections Intraventricular Neurons General Neuroscience Lentivirus HEK 293 cells Gene Transfer Techniques Brain Cell Differentiation Amniotic Fluid Flow Cytometry Immunohistochemistry Molecular biology Embryonic stem cell Bone morphogenetic protein 7 Cerebrospinal fluid Neuronal differentiation embryonic structures Female Transforming growth factor |
Zdroj: | Journal of Neuroscience Methods. 205:17-27 |
ISSN: | 0165-0270 |
Popis: | 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. |
Databáze: | OpenAIRE |
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