title: Viral transduction of primary Schwann cells using a Cre-lox system to regulate GDNF expression.
abstract: 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.
authors.affiliation: Division of Plastic and Reconstructive Surgery, Department of Surgery, Washington University School of Medicine, Campus Box 8238, 660 South Euclid Avenue, St. Louis, Missouri, 63110.
authors.forename: Yuewei
authors.initials: Y
authors.lastname: Wu-Fienberg
authors.forename: Amy M
authors.initials: AM
authors.lastname: Moore
authors.forename: Laura M
authors.initials: LM
authors.lastname: Marquardt
authors.forename: Piyaraj
authors.initials: P
authors.lastname: Newton
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Johnson
authors.forename: Susan E
authors.initials: SE
authors.lastname: Mackinnon
authors.forename: Shelly E
authors.initials: SE
authors.lastname: Sakiyama-Elbert
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Wood
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000096:biosynthesis* / Q000235:genetics; D016086:Lentivirus / Q000235:genetics; D011917:Rats, Inbred Lew; D011995:Recombination, Genetic; D012583:Schwann Cells / Q000378:metabolism*; D014161:Transduction, Genetic
chemical_list: D051100:Glial Cell Line-Derived Neurotrophic Factor
keywords: candy-store effect; cre-lox; drug delivery; glial cell line derived neurotrophic factor; lentivirus; peripheral nerve
references.citation: Nat Immunol. 2007 Jul;8(7):665-8
references.pmid: 17579640
references.citation: J Neurosci Res. 2002 Dec 15;70(6):746-55
references.pmid: 12444596
references.citation: Methods Mol Biol. 2010;655:47-64
references.pmid: 20734253
references.citation: Proc Natl Acad Sci U S A. 2006 Sep 12;103(37):13759-64
references.pmid: 16945906
references.citation: Endocrine. 2002 Dec;19(3):221-8
references.pmid: 12624421
references.citation: J Control Release. 2013 Dec 28;172(3):841-51
references.pmid: 24140746
references.citation: Nat Protoc. 2012 Nov;7(11):1996-2004
references.pmid: 23060244
references.citation: Exp Neurol. 2010 May;223(1):207-20
references.pmid: 19646436
references.citation: Plast Reconstr Surg. 2011 May;127(5):105e-118e
references.pmid: 21532404
references.citation: Neurosurgery. 2009 Oct;65(4 Suppl):A105-14
references.pmid: 19927054
references.citation: Exp Neurol. 2003 Oct;183(2):610-9
references.pmid: 14552902
references.citation: Regen Med. 2013 Jan;8(1):27-37
references.pmid: 23259803
references.citation: Muscle Nerve. 2013 Feb;47(2):213-23
references.pmid: 23169341
references.citation: Mol Cell Neurosci. 2012 May;50(1):103-12
references.pmid: 22735691
references.citation: J Neurosci. 1994 Mar;14(3 Pt 1):1309-19
references.pmid: 8120626
references.citation: Neurosci Lett. 1998 Jul 3;250(2):87-90
references.pmid: 9697925
references.citation: Exp Neurol. 2004 Oct;189(2):303-16
references.pmid: 15380481
references.citation: Annu Rev Neurosci. 1986;9:305-28
references.pmid: 3518587
references.citation: Neuroreport. 2000 Jun 5;11(8):1651-4
references.pmid: 10852218
references.citation: J Cell Biol. 1995 Jul;130(1):137-48
references.pmid: 7790368
references.citation: Gene Ther. 2006 Oct;13(19):1382-90
references.pmid: 16724096
references.citation: J Biol Chem. 2004 Apr 30;279(18):18776-82
references.pmid: 14761948
references.citation: J Biomed Mater Res A. 2009 Jun 15;89(4):909-18
references.pmid: 18465825
references.citation: Eur J Neurosci. 1997 Jul;9(7):1450-60
references.pmid: 9240402
references.citation: Neuron. 1996 Aug;17(2):353-61
references.pmid: 8780658
references.citation: J Neurosci. 1991 Aug;11(8):2433-42
references.pmid: 1869923
references.citation: Restor Neurol Neurosci. 2007;25(5-6):585-99
references.pmid: 18418947
references.citation: J Commun Disord. 2010 Jul-Aug;43(4):265-73
references.pmid: 20451212
references.citation: J Neurosci. 1992 Sep;12(9):3310-20
references.pmid: 1527582
references.citation: Mol Cell Neurosci. 2008 Sep;39(1):105-17
references.pmid: 18585464
references.citation: Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5547-51
references.pmid: 1319065
references.citation: Hum Gene Ther. 2006 Jan;17(1):1-9
references.pmid: 16409120
references.citation: Neurosci Lett. 2008 Sep 26;443(1):46-50
references.pmid: 18662744
references.citation: Science. 1995 Jun 23;268(5218):1766-9
references.pmid: 7792603
references.citation: Eur J Neurosci. 2002 Feb;15(4):589-601
references.pmid: 11886440
references.citation: J Neurosci Methods. 2011 Apr 30;197(2):209-15
references.pmid: 21354206
references.citation: PLoS One. 2013 Aug 12;8(8):e71076
references.pmid: 23951085
references.citation: Eur J Neurosci. 2008 Oct;28(8):1467-79
references.pmid: 18973572
references.citation: Exp Neurol. 2002 Jan;173(1):77-85
references.pmid: 11771940
references.citation: Biotechnol Bioeng. 2013 May;110(5):1272-81
references.pmid: 23239194
references.citation: J Neurol. 1994 Dec;242(1 Suppl 1):S19-21
references.pmid: 7699403
references.citation: J Neurosurg. 2010 Jul;113(1):102-9
references.pmid: 19943736
references.citation: J Neurotrauma. 2004 Oct;21(10):1468-78
references.pmid: 15672636
references.citation: Neurosurgery. 2009 Oct;65(4 Suppl):A132-44
references.pmid: 19927058
references.citation: Clin Plast Surg. 2011 Oct;38(4):643-60
references.pmid: 22032591