Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for <em>In Vitro</em> Myelination Assays

Abstract

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). We use an in vitro myelination assay, an established model for studying CNS myelination in vitro. To do this, oligodendrocyte precursor cells (OPCs) are added to the purified primary rodent dorsal root ganglion (DRG) neurons to form myelinating co-cultures. In order to specifically interrogate the roles that particular proteins expressed by oligodendrocytes exert upon myelination we have developed protocols that selectively transduce OPCs using the lentivirus overexpressing wild type, constitutively active or dominant negative proteins before being seeded onto the DRG neurons. This allows us to specifically interrogate the roles of these oligodendroglial proteins in regulating myelination. The protocols can also be applied in the study of other cell types, thus providing an approach that allows selective manipulation of proteins expressed by a desired cell type, such as oligodendrocytes for the targeted study of signaling and compensation mechanisms. In conclusion, combining the in vitro myelination assay with lentiviral infected OPCs provides a strategic tool for the analysis of molecular mechanisms involved in myelination. Video LinkThe video component of this article can be found at

Fyn is an intermediate kinase that BDNF utilizes to promote oligodendrocyte myelination

“…The culture of DRG neurons has been described previously (Peckham et al, ; Xiao et al, ). Briefly, DRG neurons were isolated from P2 S/D rats and plated on to polyornithine (P3655) and laminin (23017015 Life Technologies) coated coverslips.…”

“…OPCs were isolated from P7–8 S/D rat brain by sequential immunopanning as previously described (Peckham et al, ; Xiao et al, ). Briefly, cerebral hemispheres were diced and digested with papain (20 U/mL, LS003126 Worthington Biochemical Cooperation) at 37°C.…”

“…Cells were isolated through three sequential immunopanning dishes: Ran‐2 (TIB119 ATCC), anti‐O1 (MAB344 Millipore) and anti‐O4 (MAB345 Millipore). O4 + O1 − OPCs were released from the final panning dish with trypsin EDTA (25300‐054 Life Technologies) and cultured in SATO medium as previously described (Peckham et al, ).…”

“…The original kinase dead (KD)‐Fyn (K299M) and wild‐type (WT)‐Fyn plasmids (Takeuchi et al, ) were subcloned into the lentiviral plasmid 2K7 as previously described (Peckham et al, ; Xiao et al, ). Lentivirus was prepared from the final lentiviral plasmids (2K7‐CMV‐IRES‐EGFP Amp [control empty vector], 2K7‐CMV‐Flag‐KD‐Fyn‐IRES‐EGFP Amp and 2K7‐CMV‐Flag‐WT‐Fyn‐IRES‐EGFP Amp) by transfection into HEK293T cells as previously described (Peckham et al, ; Xiao et al, ). The concentrated lentiviral supernatant was titered on HEK293T cells and O4 + O1 − OPCs were infected with a virus concentration that maximized protein expression with no obvious cell death.…”

“…DRG‐OPC cocultures were established based on published techniques (Peckham et al, ; Xiao et al, ). Briefly, OPCs were seeded onto coverslips (200,000 OPCs/22‐mm coverslip) containing purified DRGs under the same approximate axon density.…”