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

Summary

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.

Abstract

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 (Ψ) 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 (http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf). 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 (http://www.addgene.org/). 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.

Protocol

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 penicillin, 100 μg/ml streptomycin in 37 °C incubator with 5% CO₂.

1. Seed 293T cells at 30-40% confluence to 10 100-mm tissue cul....

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Sigma-Aldrich	D5796
MEM	Invitrogen	11090-081
Fetal bovine serum	Hyclone	SV3001403
PBS	Mediatech, Inc.	21-040-CM
Trypsin-EDTA	Sigma-Aldrich	T3924
Sodium butyrate	Sigma-Aldrich	B5887
Hexadimethrine bromide (Polybrene)	Sigma-Aldrich	H9268
293T cells	ATCC	CRL-11268
HT1080 cells	ATCC	CCL-121
Falcon 100 x 20 mm tissue culture dish	BD Biosciences	353003
1 x 3 ½ in polyallom–r centrifuge tube	Beckman Coulter Inc.	326823
0.2-micron syringe filter	Corning	431219
QIAamp DNA Mini Kit	Qiagen	51304