This work was supported by the Australian National Health and Medical Research Council (NHMRC fellowship #454330 to JX, project grant #628761 to SM and APP1058647 to JX), Multiple Sclerosis Research Australia (MSRA #12070 to JX), the University of Melbourne Research Grant Support Scheme and Melbourne Research CI Fellowship to JX as well as Australia Postgraduate Scholarships to HP and AF.&#160;We would like to acknowledge the Operational Infrastructure Scheme of the Department of Innovation, Industry and Regional Development, Victoria Australia.

<ol><li>Baumann, N., Pham-Dinh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biology+of+oligodendrocyte+and+myelin+in+the+mammalian+central+nervous+system%5BTitle%5D)+AND+%22Physiol+Rev%22%5BJournal%5D)">Biology of oligodendrocyte and myelin in the mammalian central nervous system.</a> Physiol Rev. 81 (2), 871-927 (2001).</li>
<li>Watkins, T. A., Emery, B., Mulinyawe, S., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+stages+of+myelination+regulated+by+gamma-secretase+and+astrocytes+in+a+rapidly+myelinating+CNS+coculture+system%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Distinct stages of myelination regulated by gamma-secretase and astrocytes in a rapidly myelinating CNS coculture system.</a> Neuron. 60 (4), 555-569 (2008).</li>
<li>Lee, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MDGAs+interact+selectively+with+neuroligin-2+but+not+other+neuroligins+to+regulate+inhibitory+synapse+development%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">MDGAs interact selectively with neuroligin-2 but not other neuroligins to regulate inhibitory synapse development.</a> Proc Natl Acad Sci U S A. 110 (1), 336-341 (2013).</li>
<li>Xiao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BDNF+exerts+contrasting+effects+on+peripheral+myelination+of+NGF-dependent+and+BDNF-dependent+DRG+neurons%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">BDNF exerts contrasting effects on peripheral myelination of NGF-dependent and BDNF-dependent DRG neurons.</a> J Neurosci. 29 (13), 4016-4022 (2009).</li>
<li>Chan, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NGF+controls+axonal+receptivity+to+myelination+by+Schwann+cells+or+oligodendrocytes%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">NGF controls axonal receptivity to myelination by Schwann cells or oligodendrocytes.</a> Neuron. 43 (2), 183-191 (2004).</li>
<li>Xiao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brain-Derived+Neurotrophic+Factor+Promotes+Central+Nervous+System+Myelination+via+a+Direct+Effect+upon+Oligodendrocytes%5BTitle%5D)+AND+%22Neurosignals%22%5BJournal%5D)">Brain-Derived Neurotrophic Factor Promotes Central Nervous System Myelination via a Direct Effect upon Oligodendrocytes.</a> Neurosignals. 18 (3), 186-202 (2010).</li>
<li>Lundgaard, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuregulin+and+BDNF+induce+a+switch+to+NMDA+receptor-dependent+myelination+by+oligodendrocytes%5BTitle%5D)+AND+%22PLoS+Biology%22%5BJournal%5D)">Neuregulin and BDNF induce a switch to NMDA receptor-dependent myelination by oligodendrocytes.</a> PLoS Biology. 11 (12), e1001743(2013).</li>
<li>Kleitman, N., W, P. M., Bunge, R. P. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKleitman%2C+N+%22Tissue+culture+methodes+for+the+study+of+myelination%22">Tissue culture methodes for the study of myelination</a>. , MIT Press. Cambridge, MA. (1991).</li>
<li>Xiao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+signal-regulated+kinase+1%2F2+signaling+promotes+oligodendrocyte+myelination+in+vitro%5BTitle%5D)+AND+%22J+Neurochem%22%5BJournal%5D)">Extracellular signal-regulated kinase 1/2 signaling promotes oligodendrocyte myelination in vitro.</a> J Neurochem. 122 (6), 1167-1180 (2012).</li>
<li>Wong, A. W., Xiao, J., Kemper, D., Kilpatrick, T. J., Murray, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oligodendroglial+expression+of+TrkB+independently+regulates+myelination+and+progenitor+cell+proliferation%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%22%5BJournal%5D)">Oligodendroglial expression of TrkB independently regulates myelination and progenitor cell proliferation.</a> The Journal of Neuroscience. 33 (11), 4947-4957 (2013).</li>
<li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+cloning%2C+Characterization+and+Expression+of+miR-15a-3p+and+miR-15b-3p+in+Dairy+Cattle%5BTitle%5D)+AND+%22Molecular+and+Cellular+Probes%22%5BJournal%5D)">Molecular cloning, Characterization and Expression of miR-15a-3p and miR-15b-3p in Dairy Cattle.</a> Molecular and Cellular Probes. , (2014).</li>
<li>Emery, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myelin+gene+regulatory+factor+is+a+critical+transcriptional+regulator+required+for+CNS+myelination%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Myelin gene regulatory factor is a critical transcriptional regulator required for CNS myelination.</a> Cell. 138 (1), 172-185 (2009).</li>
<li>Murai, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nuclear+receptor+TLX+stimulates+hippocampal+neurogenesis+and+enhances+learning+and+memory+in+a+transgenic+mouse+model%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Nuclear receptor TLX stimulates hippocampal neurogenesis and enhances learning and memory in a transgenic mouse model.</a> Proc Natl Acad Sci U S A. 111 (25), 9115-9120 (2014).</li>
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The authors declare that there is no conflict of interest regarding this research.

Myelination of axons is a crucial process for the optimal function of both the central and peripheral nervous systems of vertebrates. The generation and maintenance of myelinated axons is a complex and coordinated process involving molecular interactions between neuronal, glial (from Schwann cells or oligodendrocytes) and extra cellular matrix proteins. The significance and applicability of this protocol is that it allows manipulation of proteins in one specific cell type within the mixed co-culture settings. As multiple cell types are involved in myelination both in vivo and in the in vitro myelination assay, using blunt pharmacological tools to block the activity of particular proteins or signaling pathways cannot provide specific information about the influence that proteins exert upon myelination from either a neuronal or glial perspective, unless the protein is uniquely expressed by one cell type. Thus, the in vitro myelination assay in conjunction with the use of Lentivirus to specifically transduce OPCs allows us to interrogate the effects that oligodendroglial proteins exert upon myelination. We have successfully used 2K7 Lentivirus to overexpress particular proteins specifically in oligodendrocytes for the in vitro myelination assay in order to dissect the effect that oligodendroglial signals exert upon CNS myelination9.
To achieve reproducible results in the transduced OPC in vitro myelination assay, optimization is required for each step of the protocol from cloning through dissections and OPC seeding. It is imperative that, at the level of DNA, the pENTR vector containing the tagged gene of interest construct is sequenced and that the 2K7 DNA with the tagged gene of interest is checked by western blot for expression of the tag and the gene of interest, prior to using the DNA to generate virus. It is essential to choose either HEK293Tcells or HEK293 FT cells to generate the virus and grow under mycoplasma free environment. After producing lentivirus it is important to titrate each batch of virus to determine the optimum dilution to use on OPCs. This can be done by adding a range of viral dilutions to HEK293T cells or to OPCs. As the 2K7 construct contains both the gene-of-interest and GFP, efficacy of transduction can be assessed by expression of GFP by fluorescence microscopy and by assaying expression of the tag and the gene of interest by western blot analysis. Brighter GFP+ cells will become visible with higher virus concentrations, as it is possible for multiple viral particles to infect a single cell. Verification of the expression of GFP via fluorescence microscopy is a helpful indicator of titer&#160;of virus and expression of GFP but cannot be used as a surrogate for checking the expression of the protein of interest. It is therefore critical to verify the expression level of the protein of interest in both HEK293T cells and OPCs by western blot analysis. If the protein of interest is expressed endogenously by neurons in the co-cultures, the overexpression by OPCs/OLs may be masked when analyzing the co-culture lysate by western blot; thus it is important to either tag the protein of interest or culture the sister OPCs following seeding so expression of the protein of interest can be verified in the sister OPCs if not in the co-culture.
The quality of both DRG and OPC culture directly determines if the experiments succeeds. In the coculture settings, rat DRG neurons can be cocultured with OPCs derived from either rat or mouse. OPCs require gentle and swift handling. It is important to optimize the virus titer&#160;for OPCs as too little fails to effectively transduce the OPCs leading to low levels of expression of the protein of interest and too much virus is toxic to OPCs so there are too few cells to seed onto the neurons. Both of these sub optimal transductions may result in unanalyzable or insignificant changes in myelination or myelin protein expression. OPCs differentiate if they become over confluent and cannot be seeded after they have differentiated, thus the number of OPCs cultured and transduced needs to be optimized to local conditions. It is desirable to seed the same number of OPCs for each myelination assay allowing direct comparisons of the number of myelinated axonal segments to be compared between conditions over multiple experiments.
A potential limitation of this technique is the variability in basal myelination levels between myelination assays. Following viral transduction, the basal level of myelination is reduced, suggesting that virally infected OPCs do not myelinate as well as the naive (non-viral infected) OPCs. This can be overcome by quantifying the extent of myelination relative to the basal myelination in each assay. Thus, the empty vector that expresses only GFP must be used as an internal control. In addition to myelination, the expression of a protein of interest may also influence other aspects of OPCs such as survival, proliferation and differentiation, leading to an indirect effect on myelination. Therefore, analysis of OPC behavior such as cell viability should be undertaken in parallel to myelination assays.
The viral transduction methods described here are not limited to the study of oligodendrocyte myelination; in fact it can be generalized and applied to study other cell types such as Schwann cells, astrocytes, and neurons, while optimization may be required for individual cell type. This protocol offers great flexibility to study cell behavior such as proliferation and differentiation by selective overexpression of a protein of interest (wild type or mutant) in the desired cell type, which has particular advantages when using a mixed cell culture system. The cells that are used for transduction can be isolated from either rat or mouse (wild type or transgenic), thus allowing for the targeted study of signaling and compensation mechanisms in transgenic animals, which is usually difficult to achieve and much more time consuming than using an in vivo transgenic mouse model. Recent evidence suggest that the lentiviral-based strategy has been successfully used for cell transduction in animal models13, suggesting a strong potential of transducing oligodendroglial cells using the lentiviral approaches in vivo.
In conclusion, combining the in vitro myelination assay with Lentiviral infection of OPCs provides a strategic tool for the analysis of molecular mechanisms involved in myelination. The role of specific proteins in myelination can be interrogated and as OPCs can be isolated from rats and mice for use on rat DRG co-cultures, the lentiviral approach can also be used in combination with knockout technology of various mouse lines to interrogate mechanisms of compensation in the processes of myelination.

Myelination of axons is crucial for the fast and efficient transmission of action potentials in both the central and peripheral nervous systems. Specialized cells, Schwann cells in the peripheral nervous system and oligodendrocytes in the central nervous system, wrap around and ensheathe axons in myelin, effectively insulating the nerve and facilitating saltatory conduction1. The process of myelination can be studied in vitro using retinal ganglion neurons2, engineered nanofibers3, or dorsal root ganglion neurons co-cultured with either Schwann cells4 or oligodendrocytes5-7. The in vitro myelination assay is an established model for studying nervous system myelination and it replicates many of the fundamental processes that occur during myelination in vivo5-8. The assay involves the coculture of purified populations of Dorsal Root Ganglion (DRG) neurons, with OPCs (for CNS myelination) or Schwann cells (for PNS myelination). Under specific conditions these myelinating cells ensheathe DRG axons in the ordered, ultra structurally verified, multi-lamellar sheet of insulating plasma membrane that express the same complement of myelin specific proteins present in vivo.
The most commonly used cell model of studying CNS myelination in vitro is the co-cultures of DRG neurons and OPCs, which have been successfully used to study the effect that exogenous factors such as the neurotrophins exert on CNS myelination in vitro5,6. Exogenous factors such as growth factors or small molecule pharmacological inhibitors have been widely used to study the role of signaling pathways in myelination using the DRG-OPC coculture model7,9. However, in the mixed co-culture settings that contain both the neurons and oligodendrocytes, it remained formally possible that either the growth factors or the pharmacological inhibitors could have exerted effects upon both the DRG neurons and oligodendrocytes (OL). This does offer the ability to specifically dissect the roles that the proteins expressed only by DRGs or oligodendroglia exerts upon myelination using this dual cell system. To unequivocally confirm that the signaling pathway in oligodendroglial directly regulates myelination, lentiviral transduction of OPCs, prior to seeding onto DRG neurons for the in vitro myelination assay, has proven to be an elegant way to overexpress both wild-type and mutant proteins, as well as knockdown expression of constitutively expressed proteins by oligodendrocytes. Thus this approach offers an avenue to specifically interrogate and manipulate signaling pathways within oligodendrocytes for studying myelination9,10.
In this paper, we report methods that we have developed to overexpress a protein of interest selectively in oligodendrocytes via a lentiviral approach for studying myelination in vitro. The technique begins with the generation of expression vectors containing the gene of interest, be it in a wild type, constitutively active or dominant negative form which are then subsequently cloned into the pENTR vector (pENTR L1-L2 pENTR4IRES2GFP). This vector (containing the gene of interest), the CMV promotor donor (pENTR L4-R1 pENTR-pDNOR-CMV) and the 2K7 lentivector are combined in an enzyme reaction to produce a 2K7 vector containing CMV promoter, the gene of interest, an internal ribosomal entry site and GFP (Figure 1). This Gateway cloned 2K7 construct combined with the PMD2.G virus envelope and the pBR8.91 virus package can be co-transfected into HEK293T cells to generate lentivirus that can subsequently be used to transduce OPCs. Once infected with the lentivirus the OPCs express a high level of the protein of interest. These OPCs can then be seeded onto DRG neuron cultures and the effect that expression of high levels of the desired protein exerts on myelination can be interrogated. The co-cultures are assessed for myelin protein expression by western blot analysis and visualized for the formation of myelinated axonal segments by immunocytochemistry.

<table><tbody><tr><td>2K7 lentivector&amp;nbsp;</td><td></td><td></td><td>Kind gift from Dr Suter&lt;sup&gt;9&lt;/sup&gt;</td></tr><tr><td>5-Fluoro-2&amp;prime;-deoxyuridine</td><td>Sigma-Aldrich</td><td>F0503-100mg</td><td></td></tr><tr><td>Alexa Fluor 488 Goat anti-mouse IgG</td><td>Jackson Immunoresearch</td><td>115545205</td><td></td></tr><tr><td>Alexa Fluor 488 goat anti-rabbit IgG (H+L)</td><td>Life Technologies</td><td>A11008</td><td></td></tr><tr><td>Alexa Fluor 594 goat anti-mouse IgG (H+L)</td><td>Life Technologies</td><td>A11005</td><td></td></tr><tr><td>Alexa Fluor 594 goat anti-rabbit IgG (H+L)</td><td>Life Technologies</td><td>A11012</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma-Aldrich</td><td>A9518-5G</td><td></td></tr><tr><td>B27 - NeuroCul SM1 Neuronal Supplement</td><td>Stem Cell Technologies&amp;nbsp;</td><td>5711</td><td></td></tr><tr><td>BDNF (Human)</td><td>Peprotech</td><td>PT450021000</td><td></td></tr><tr><td>Biotin (d-Biotin)</td><td>Sigma Aldrich</td><td>B4639</td><td></td></tr><tr><td>Bradford Reagent</td><td>Sigma Aldrich</td><td>B6916-500ML&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A4161</td><td></td></tr><tr><td>Chloramphenicol</td><td>Sigma-Aldrich</td><td>C0378-100G</td><td></td></tr><tr><td>CNTF</td><td>Peprotech</td><td>450-13020</td><td></td></tr><tr><td>DAKO fluoresence mounting media</td><td>DAKO</td><td>S302380-2</td><td></td></tr><tr><td>DMEM, high glucose, pyruvate, no glutamine</td><td>Life Technologies</td><td>10313039</td><td></td></tr><tr><td>DNase</td><td>Sigma-Aldrich</td><td>D5025-375KU</td><td></td></tr><tr><td>DPBS</td><td>Life Technologies</td><td>14190250</td><td></td></tr><tr><td>DPBS, calcium, magnesium</td><td>Life Technologies</td><td>14040182</td><td></td></tr><tr><td>EBSS</td><td>Life Technologies</td><td>14155063</td><td></td></tr><tr><td>EcoRI-HF</td><td>NEB</td><td>R3101</td><td></td></tr><tr><td>Entry vectors for promoter and gene of interest</td><td></td><td></td><td>Generate as per protocols 1-2</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>12003C</td><td></td></tr><tr><td>Forskolin&amp;nbsp;</td><td>Sigma Aldrich</td><td>F6886-50MG</td><td></td></tr><tr><td>Glucose (D-glucose)</td><td>Sigma-Aldrich</td><td>G7528</td><td></td></tr><tr><td>Glycerol</td><td>Chem Supply</td><td>GL010-500M</td><td>See stock solutions</td></tr><tr><td>Goat Anti-Mouse IgG</td><td>Jackson ImmunoResearch</td><td>115005003</td><td></td></tr><tr><td>Goat Anti-Mouse IgM&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>115005020</td><td></td></tr><tr><td>Goat Anti-Rat IgG</td><td>Jackson ImmunoResearch</td><td>112005167</td><td></td></tr><tr><td>Hoechst 33342</td><td>Life Technologies</td><td>H3570</td><td></td></tr><tr><td>Igepal</td><td>Sigma Aldrich</td><td>I3021-100ML&amp;nbsp;</td><td></td></tr><tr><td>Insulin&amp;nbsp;</td><td>Sigma Aldrich</td><td>I6634&amp;nbsp;</td><td></td></tr><tr><td>Kanamycin</td><td>Sigma-Aldrich</td><td>60615</td><td></td></tr><tr><td>Laminin&amp;nbsp;</td><td>Life Technologies</td><td>23017015</td><td></td></tr><tr><td>LB Medium</td><td></td><td></td><td>See stock solutions</td></tr><tr><td>LB-Agar</td><td></td><td></td><td>See stock solutions</td></tr><tr><td>L-Cysteine</td><td>Sigma-Aldrich</td><td>C-7477</td><td></td></tr><tr><td>Leibovitz&#039;s L-15 Medium</td><td>Life Technologies</td><td>11415064</td><td></td></tr><tr><td>L-Glutamate</td><td>Sigma-Aldrich</td><td>G1626</td><td></td></tr><tr><td>L-Glutamine- 200 mM (100x) liquid</td><td>Life Technologies</td><td>25030081</td><td></td></tr><tr><td>LR Clonase II Plus enzyme</td><td>Life Technologies</td><td>12538-120</td><td></td></tr><tr><td>MEM, NEAA, no Glutamine</td><td>Life Technologies</td><td>10370088</td><td></td></tr><tr><td>Mouse &amp;alpha; 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neurofilament</td><td>Millipore</td><td>AB1987&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Rabbit &amp;alpha;MBP (polyclonal)</td><td>Millipore</td><td>AB980</td><td></td></tr><tr><td>Ran2 hybridoma cells</td><td>ATCC</td><td>TIB-119</td><td>Conditioned medium containing anti-Ran2 antibody to be used for immunopanning</td></tr><tr><td>Rat anti CD140A/PDGFRa antibody</td><td>BD Pharmingen</td><td>558774</td><td></td></tr><tr><td>SacII</td><td>NEB</td><td>R0157</td><td></td></tr><tr><td>SOC medium</td><td></td><td></td><td>Supplied with competent bacteria</td></tr><tr><td>Sodium selenite&amp;nbsp;</td><td>Sigma Aldrich</td><td>S5261</td><td></td></tr><tr><td>Spe I</td><td>NEB</td><td>R0133S</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>NEB</td><td>M0202S</td><td></td></tr><tr><td>T4 DNA Ligase Buffer</td><td>NEB</td><td>B0202S</td><td></td></tr><tr><td>TE buffer pH8</td><td></td><td></td><td>See stock solutions</td></tr><tr><td>TNE lysis buffer</td><td></td><td></td><td></td></tr><tr><td>Trace Elements B</td><td>Cellgro&amp;nbsp;</td><td>99-175-CI</td><td></td></tr><tr><td>Transferrin (apo-Transferrin human)</td><td>Sigma-Aldrich</td><td>T1147</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284&amp;nbsp;</td><td></td></tr><tr><td>Trypsin</td><td>Sigma-Aldrich</td><td>T9201-1G</td><td></td></tr><tr><td>Trypsin Inhibitor From Chicken Egg White</td><td>Roche</td><td>10109878001</td><td></td></tr><tr><td>Trypsin-EDTA (1x), phenol red (0.05%)</td><td>Life Technologies</td><td>25300-054</td><td></td></tr><tr><td>Unconjugated Griffonia Simplicifolia Lectin BSL-1</td><td>Vector laboratories&amp;nbsp;</td><td>L-1100</td><td></td></tr><tr><td>Uridine</td><td>Sigma-Aldrich</td><td>U3003-5G</td><td></td></tr></tbody></table>

production and use of lentivirus to selectively transduce primary oligodendrocyte precursor cells for in vitro myelination assays

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.

The flag-tagged extracellular signal-related kinase 1(Flag-Erk1) construct used for lentivirus production is verified by restriction enzyme digest of the constructs used, including both the 2K7 constructs and the packaging and accessory constructs required for virus production (Figure 3).
<img alt="Figure 3" src="/files/ftp_upload/52179/52179fig3highres.jpg" /> 
Figure 3: DNA construct verification. All DNA constructs required for lentivirus production were purified from Stbl3 bacteria and verified by restriction enzyme digest. Accessory and packaging vectors were digested with NcoI and EcoRI, as indicated, and compared to uncut plasmid (A). 2K7-GFP was digested with SpeI and SacII (A). 2K7-Flag-Erk1 DNA was verified by digestion with either SpeI or SacII alone, or by double digest (B). Banding patterns were compared to expected patterns generated from virtual digestion of the construct sequences using ApE software.
To determine the optimum dilution to use on OPCs, the Erk 1 lentivirus was titrated in HEK293T cells (Figure 4). This was done by adding a range of viral dilutions to HEK293T cells (Figures 4A, 4C, and 4D) or to OPCs (Figure 4B). Expression levels of the gene-of-interest increase with time (Figure 4D). <a href="https://www.jove.com/files/ftp_upload/52179/52179fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52179/52179fig4highres.jpg" /> 
Figure 4: Titration of Flag-Erk1 virus in HEK293T cells and OPCs. Flag-Erk1 virus was titrated in HEK293T cells (A, C, D) or OPCs (B). HEK293T cells (A) or OPCs (B) were imaged for GFP expression 48 hr after infection with Flag-Erk1 virus. HEK293T cell lysates were probed for the presence of Erk1 or total Erk1/2 after 48 hr of infection (C). HEK293T cell lysates were probed for the presence of Flag, Erk1/2 and Actin as a loading control either 48 hr or 96 hr after infection with Flag-Erk1 virus (D). These blots were all blotted and imaged together to facilitate direct comparison of expression levels at the two time points (D). Scale bar for (A) = 100 &#956;m. Scale bar for (B) = 50 &#956;m. <a href="https://www.jove.com/files/ftp_upload/52179/52179fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Once the optimal virus dilution has been determined, purified OPCs were infected and cultured for a minimum of 48 h to allow transgene expression to reach sufficient levels, then seeded onto DRG neurons to assess their myelination potential (Figure 5). Once seeded onto DRG neurons, OPCs will differentiate into oligodendrocytes, and begin to express myelin proteins, which can be assessed by western blotting (Figure 5C). Some mature oligodendrocytes will myelinate, and this can be visualized through MBP staining (Figure 5D). Axons also need to be stained with a marker such as Neurofilament or &#946;III-tubulin (Figure 5D) to ensure axon density is taken into account when analyzing myelination levels.
<img alt="Figure 5" src="/files/ftp_upload/52179/52179fig5highres.jpg" /> 
Figure 5: OPCs were infected with Flag-Erk1 virus for 48 hr before seeding onto DRG neurons. Sister OPCs were either fixed and stained for Flag and GFP (A) or lysed and probed for Flag and Erk1 to verify expression of the virally-overexpressed protein (C). Co-cultures were fixed after 2 weeks in culture and stained for &#946;III-tubulin and MBP to visualize DRG axons and myelinating as well as non-myelinating oligodendrocytes (B). A myelinating oligodendrocyte is indicated by the arrow and a non-myelinating oligodendrocyte is indicated by the arrow-head (B). Parallel co-cultures were lysed and probed for the myelin proteins MBP and MAG, as well as Flag and Erk1/2 to confirm transgene expression in the co-culture (D). An increase in Erk1 levels was not visible in the co-culture due to the large quantities of DRG-derived Erk1 present in the lysates Scale bar = 20 &#956;m.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Ingredients</td>
			<td>Notes</td>
		</tr>
		<tr>
			<td>TE Buffer pH 8</td>
			<td>10 mM Tris pH 8 
			1 mM EDTA pH 8</td>
			<td>Make up in deionized water.</td>
		</tr>
		<tr>
			<td>Polyethylenimine (PEI)</td>
			<td>1 g/L</td>
			<td>Make up in deionized water, filter-sterilize and store stocks at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>LB Medium</td>
			<td>20 g Tryptone 
			10 g Yeast extract 
			10 g NaCl</td>
			<td>Make up to 2 L with deionized water</td>
		</tr>
		<tr>
			<td>50% Glycerol stock</td>
			<td>Glycerol</td>
			<td>Make up with an equal volume of deionized water and autoclave</td>
		</tr>
		<tr>
			<td>HEK 293 T cell medium</td>
			<td>DMEM 
			10% Fetal Bovine Serum 
			1% Penicillin/streptomycin 
			1% L-glutamine</td>
			<td></td>
		</tr>
		<tr>
			<td>TNE lysis buffer</td>
			<td>10 mM Tris pH 8 
			150 mM NaCl 
			1 mM EDTA pH 8 
			1% NP40</td>
			<td>Dissolve NP40 in a smaller volume of deionized water first as it will crystallize on contact with water. Make up to final volume in deionized water</td>
		</tr>
		<tr>
			<td>M1</td>
			<td>MEM 
			10% Fetal Bovine Serum 
			0.4% D-glucose 
			2 nM L-glutamate 
			1% Penicillin/streptomycin</td>
			<td>For use with rat DRG neurons</td>
		</tr>
		<tr>
			<td>mM1</td>
			<td>Neurobasal medium 
			2% B27 (SM1) supplement 
			0.4% D-glucose 
			2 nM L-glutamate 
			1% Penicillin/streptomycin</td>
			<td>For use with mouse DRG neurons</td>
		</tr>
		<tr>
			<td>M2</td>
			<td>DMEM 
			10 mg/L Transferrin 
			5 mg/L Insulin 
			20 nM Progesterone 
			100 &#956;M Putrescine 
			10 &#956;M FdU 
			10 &#956;M Uridine</td>
			<td>Make up M2 in a larger volume DMEM without FdU and uridine for use over 1-2 months. Add FdU and uridine to smaller volumes that can be finished within 2 weeks</td>
		</tr>
		<tr>
			<td>mM2</td>
			<td>mM1 
			10 &#956;M FdU 
			10 &#956;M Uridine</td>
			<td>Add FdU and uridine to smaller volumes of medium that can be finished within 2 weeks.</td>
		</tr>
		<tr>
			<td>10x MT-PBS pH 7.4</td>
			<td>28.48 g/L (160 mM) Na2HPO4&#183;2H2O 
			5.52 g/L (40 mM) NaH2PO4&#183;H2O 
			87.66 g/L (1.5 M) NaCl</td>
			<td>Add to deionized water and adjust pH to 7.4 with 10 N NaOH</td>
		</tr>
		<tr>
			<td>1x MT-PBS</td>
			<td>10x MT-PBS 
			Deionized water</td>
			<td>Dilute 10x MT-PBS 1:10 in deionized water to make 1x MT-PBS</td>
		</tr>
		<tr>
			<td>10x Borate buffer (1.5 M)</td>
			<td>18.55 g Boric acid 
			1x MT-PBS</td>
			<td>Dissolve boric acid in 150 ml 1x MT-PBS. Adjust to pH 8.56 with 10 N NaOH and bring final volume to 200 ml with 1x MT-PBS. Autoclave</td>
		</tr>
		<tr>
			<td>1x Borate buffer (0.15 M)</td>
			<td>10x Borate buffer 
			1x MT-PBS</td>
			<td>Prepare 100 ml of this buffer to dissolve PLN in. Add 10 ml of 10x borate buffer (pH 8.56) to 90 ml of 1x MT-PBS</td>
		</tr>
		<tr>
			<td>0.5 mg/ml Poly-L-ornithine (PLN)</td>
			<td>50 mg Poly-L-ornithine Hydrobromide 
			0.15 M Borate buffer (pH 8.56)</td>
			<td>Dissolve 50 mg of poly-L-ornithine hydrobromide in 100 ml of 1x borate buffer. Filter sterilize (0.22 &#956;m filter) and store at 4 &#176;C for up to one month</td>
		</tr>
		<tr>
			<td>100x Poly-D-lysine (PDL)</td>
			<td>5 mg Poly-D-lysine 
			Sterile, deionized water</td>
			<td>100x PDL stocks can be frozen at -20 &#176;C in single-use aliquots. Upon use, dilute to 1x in sterile, deionized water</td>
		</tr>
		<tr>
			<td>Papain buffer</td>
			<td>See Table 2</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>12,500 U DNase I 
			1 ml EBSS</td>
			<td>On ice, dissolve the Dnase I in 1 ml of chilled EBSS. Aliquot (e.g., 300 &#956;l/tube) and freeze overnight at -20 &#176;C. Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>4% BSA</td>
			<td>8 g BSA 
			200 ml DPBS</td>
			<td>Dissolve the BSA in 150 ml DPBS at 37 &#176;C. Adjust the pH to 7.4 with ~1 ml of 1 N NaOH. Bring the volume to 200 ml. Filter through a 0.22 &#956;m filter to sterilize. Make 1 ml aliquots and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>10x Lo Ovomucoid</td>
			<td>3 g BSA 
			200 ml DPBS 
			3 g Trypsin inhibitor</td>
			<td>Add BSA to 150 ml DPBS and mix well. Add trypsin inhibitor and mix to dissolve. Add ~1 ml of 1 N NaOH to adjust the pH to 7.4. Bring the volume to 200 ml with DPBS. Filter-sterilize through a 0.22 &#956;m filter. Make 1 ml aliquots and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>6x Hi Ovomucoid</td>
			<td>6 g BSA 
			200 ml DPBS 
			6 g trypsin inhibitor</td>
			<td>Add BSA to 150 ml DPBS and mix well. Add trypsin inhibitor and mix to dissolve. Add ~1 ml of 1N NaOH to adjust the pH to 7.4. Bring the volume to 200 ml with DPBS. Filter-sterilize through a 0.22 &#956;m filter. Make 1 m aliquots and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>SATO base</td>
			<td>See Table 3</td>
			<td></td>
		</tr>
		<tr>
			<td>SATO media (rat)</td>
			<td>1% SATO base 
			1% Penicillin/streptomycin 
			1% 0.5 mg/ml Insulin 
			1% L-Glutamine 
			0.1% NAC 
			0.1% Biotin 
			Make up with DMEM and filter sterilize.</td>
			<td></td>
		</tr>
		<tr>
			<td>SATO media (mouse)</td>
			<td>1% SATO base 
			1% 0.5 mg/ml insulin 
			1% Penicillin/streptomycin 
			1% L-Glutamine 
			0.1% NAC 
			0.1% Biotin 
			0.1% Trace Elements B 
			2% B27 
			Make up with DMEM and filter sterilize.</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>10 mg Insulin 
			20 ml Sterile deionzed water</td>
			<td>Add insulin to deionized water and add 100 &#956;l of 1 N HCl to allow the insulin to dissolve. Mix well. Filter through a 0.22 &#956;m filter and store at 4 &#176;C for 4-6 weeks</td>
		</tr>
		<tr>
			<td>NAC (N-Acetyl-L-cysteine)</td>
			<td>5 mg/ml NAC 
			DMEM</td>
			<td>Dissolve NAC in DMEM to make a 5 mg/ml solution, aliquot and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>d-Biotin</td>
			<td>50 &#956;g/ml biotin 
			Sterile, deionized water</td>
			<td>Dissolve biotin in water to make a 50 &#956;g/ml solution, aliquot and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>CNTF (ciliary neurotrophic factor)</td>
			<td>10 &#956;g/ml CNTF 
			0.2% BSA in DPBS</td>
			<td>Dilute CNTF to make a 10 &#956;g/ml solution with sterile 0.2% BSA in DPBS. Aliquot, flash freeze in liquid nitrogen and store at -80 &#176;C.</td>
		</tr>
		<tr>
			<td>PDGF (platelet derived growth factor)</td>
			<td>10 &#181;g/ml PDGF 
			0.2% BSA in DPBS</td>
			<td>Dilute PDGF master stock (prepared according to manufacturer&#8217;s instructions) to 10 &#181;g/ml with sterile 0.2% BSA in DPBS. Aliquot, flash freeze in liquid nitrogen and store at -80 &#176;C.</td>
		</tr>
		<tr>
			<td>NT3 (Neurotrophin-3)</td>
			<td>1 &#181;g/ml NT-3 
			0.2% BSA in DPBS</td>
			<td>Dilute NT3 master stock (prepared according to manufacturer&#8217;s instructions) to 1 &#181;g/ml with sterile 0.2%BSA in DPBS. Aliquot, flash freeze in liquid nitrogen and store at -80 &#176;C.</td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>50 mg Forskolin 
			Sterile DMSO</td>
			<td>Add 1 ml sterile DMSO to the 50 mg bottle of forskolin and mix well to resuspend fully. Transfer to 15 ml tube and add 11 ml sterile DMSO to reach a concentration of 4.2 mg/ml. Aliquot and store at -20 &#176;C.</td>
		</tr>
	</tbody>
</table>
Table 3. Stock solutions.

Watch this Scientific Journal Video about Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays at JoVE.com

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

Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

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

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

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Universidad Científica del Sur

Research

JoVE Journal

Developmental Biology

14.4K Views.  The University of Melbourne. Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

Video: Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating diseases such as multiple sclerosis and transverse myelitis. Myelination is required to not only facilitate rapid impulse propagation within the central nervous system, but also to provide trophic support to underlying axons. Oligodendrocyte progenitor cells (OPCs) can be studied in vitro to help identify factors that may promote or inhibit oligodendrocyte differentiation. To date, many of the methods available to evaluate this process have either required large numbers of cells, thus limiting the number of conditions that can be investigated at any one time, or labor-intensive methods of quantification. Herein, we describe a protocol for the isolation of large numbers of highly pure OPCs together with a fast and reliable method to determine oligodendrogenesis from multiple conditions simultaneously. OPCs are isolated from P5-P7 neonatal rat cortices and grown in vitro for three days prior to differentiation. Four days after differentiation, oligodendrogenesis is evaluated using a dual-infrared fluorescence-scanning assay to determine expression of the myelin protein.

Oligodendrocytes are the myelinating cells of the central nervous system. This protocol describes a method for the isolation and culture of their precursors, oligodendrocyte progenitor cells, from rat cortices, as well as a fast and reliable quantitative method to evaluate oligodendrogenesis in vitro in response to experimental factors.

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating ...

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

Genetics

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Lentiviral vectors (LVs) are a powerful means of delivering genetic material to many types of cells. Because of safety concerns associated with these HIV-1 derived vectors, producing large quantities of LVs is challenging. In this paper, we report a method for producing high titers of self-inactivating LVs. We retrovirally transduce the tet-off stable producer cell line GPR to generate a cell line, GPRS, which can express all the viral components, including a dendritic cell-specific glycoprotein, SVGmu. Then, we use concatemeric DNA transfection to transfect the LV transfer plasmid encoding a reporter gene GFP in combination with a selectable marker. Several of the resulting clones can produce LV at a titer 10-fold greater than what we achieve with transient transfection. Plus, these viruses efficiently transduce dendritic cells in vitro and generate a strong T cell immune response to our reporter antigen. This method may be a good option for producing strong LV-based vaccines for clinical studies of cancer or infectious diseases.

Here, we use retroviral transduction and concatemeric transfection to create a cell line that can express the components of a lentiviral vector (LV) in the absence of tetracycline. This LV encodes GFP and is pseudotyped with a glycoprotein, SVGmu, which is specific for a receptor on dendritic cells.

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

Immunology and Infection

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Neuroscience

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Biology

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture.

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of primary human bronchial epithelial cells.

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of ...

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

Summary

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Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

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Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells