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Viral transduction of primary Schwann cells using a Cre -lox system to regulate GDNF expression

B&B Daniel I.C.
Yuewei Wu-Fienberg1, Amy M. Moore1, Laura M. Marquardt1,2, Piyaraj Newton1, Philip J. Johnson1, Susan E. Mackinnon1, Shelly E. Sakiyama-Elbert1,2 andMatthew D. Wood1,*DOI: 10.1002/bit.
© 2014 Wiley Periodicals, Inc.
Accepted Article (Accepted, unedited articles published online and citable. The final edited and typeset version of record will appear in future.
1Division of Plastic and Reconstructive Surgery, Department of Surgery, Washington University School of Medicine, St. Louis, MO2Department of Biomedical Engineering, Washington University, St. Louis, MO* Corresponding author: Matthew D. Wood, Ph.D.
Division of Plastic and Reconstructive Surgery, Washington University School of Medicine, 660 South Euclid, Campus Box 8238, St. Louis, MO.
E-mail address: woodmd@wustl.
†This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/bit.
Glial cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor known to enhance motor nerve regeneration following its delivery. However, recent studies have determined that extended GDNF delivery to regenerating axons can entrap motor axons at the site of GDNF delivery. This entrapment leads to reduced motor axons available to reinnervate muscle. To address this issue, we designed a cell-based GDNF expression system that can temporally regulate protein expression using an inducible gene excision mechanism to prevent entrapment at the site of expression. To design this system for regulation of GDNF expression, we transduced two lentiviral vectors, one containing a constitutively active GDNF transgene flanked by two loxP sites, and the other containing a tetracycline-inducible cre transgene along with its constitutively active transactivator, into Schwann cells (SCs). These SCs over-express GDNF, but expression can be suppressed through the administration of tetracycline family antibiotics, such as doxycycline. The engineered SCs produced significantly more GDNF as compared to untransduced controls, as measured by enzyme-linked immunosorbent assay (ELISA). Following doxycycline treatment, these SCs produced significantly lower levels of GDNF and induced less neurite extension as compared to untreated SCs. Engineered SCs treated with doxycycline showed a marked increase in Cre recombinase expression, as visualized by immunohistochemistry (IHC), providing evidence of a mechanism for the observed changes in GDNF expression levels and biological activity. This cell-based GDNF expression system could have potential for future in vivo studies to provide a temporally-controlled GDNF source to promote axon growth. Biotechnol. Bioeng. © 2014 Wiley Periodicals, Inc.Get PDF (698K)More content like thisFind more content: like this articleFind more content written by:Yuewei Wu-FienbergAmy M. MooreLaura M. MarquardtPiyaraj NewtonPhilip J. JohnsonSusan E. MackinnonShelly E. Sakiyama-ElbertMatthew D.
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