This work was supported by the NIH Neuroscience Blueprint Core grant (P30 NS057105, BJS) to Washington University, Program Project Grant NS032636 (BJS) and by the Hope Center for Neurological Disorders.

1. Packaging of Lentiviral Vectors
Lentiviral vectors are produced by cotransfection of a lentiviral transfer vector and other plasmids required for packaging into 293T cells by calcium phosphate transfection method. We use 10 100-mm tissue culture dishes in this protocol. It can be scaled up or down depending on applications. The 293T cell line is maintained in Dulbecco's modified Eagles medium (DMEM) with high glucose (4500 mg/L), supplemented with 10% fetal bovine serum (FBS), 100 units/ml pe...

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No conflicts of interest declared.

In this protocol, we have shown the production of lentiviral vectors and application of these vectors in neocortical cultures. We demonstrated efficient and cell type-specific transduction with the vectors produced by these methods. When the synapsin promoter is used, GFP expression is strictly neuron specific. When the GFAP promoter is used, GFP expression is exclusively in astrocytes. If no cell type-specific expression is required, a ubiquitous promoter may be used. We found both ubiqutin and phosphoglycerate k...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Sigma-Aldrich</td>
 <td>D5796</td>
 </tr>
 <tr>
 <td>MEM</td>
 <td>Invitrogen</td>
 <td>11090-081</td>
 </tr>
 <tr>
 <td>Fetal bovine serum</td>
 <td>Hyclone</td>
 <td>SV3001403</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Mediatech</td>
 <td>21-040-CM</td>
 </tr>
 <tr>
 <td>Trypsin-EDTA</td>
 <td>Sigma-Aldrich</td>
 <td>T3924</td>
 </tr>
 <tr>
 <td>Sodium butyrate</td>
 <td>Sigma-Aldrich</td>
 <td>B5887</td>
 </tr>
 <tr>
 <td>Hexadimethrine bromide (Polybrene)</td>
 <td>Sigma-Aldrich</td>
 <td>H9268</td>
 </tr>
 <tr>
 <td>293T cells</td>
 <td>ATCC</td>
 <td>CRL-11268</td>
 </tr>
 <tr>
 <td>HT1080 cells</td>
 <td>ATCC</td>
 <td>CCL-121</td>
 </tr>
 <tr>
 <td>Falcon 100 x 20 mm tissue culture dish</td>
 <td>BD Biosciences</td>
 <td>353003</td>
 </tr>
 <tr>
 <td>1 x 3 &frac12; in polyallomoer centrifuge tube</td>
 <td>Beckman-Coulter </td>
 <td>326823 </td>
 </tr>
 <tr>
 <td>0.2-micron syringe filter</td>
 <td>Corning</td>
 <td>431219</td>
 </tr>
 <tr>
 <td>QIAamp DNA Mini Kit</td>
 <td>Qiagen</td>
 <td>51304</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System at JoVE.com

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

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 ...

production-lentiviral-vectors-for-transducing-cells-from-central

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Neuroscience

35.7K visualizações.  Washington University School of Medicine. Neste protocolo descrevemos produção, purificação e titulação de lentivirais. Nós fornecemos um exemplo de entrega do gene lentiviral vetor mediada em primárias neurônios cultivados e astrócitos. Nossos métodos podem também se aplicam a outros tipos de células In vitro E In vivo.