Elsevier

Methods in Enzymology

Volume 406, 2006, Pages 593-605
Methods in Enzymology

Lentiviral Delivery of RNAi in Hippocampal Neurons

https://doi.org/10.1016/S0076-6879(06)06046-0Get rights and content

Abstract

The breakthrough discovery that double‐stranded RNA of 21 nucleotides in length (referred to as short or small interfering RNA; siRNA) can trigger sequence‐specific gene silencing in mammalian cells has led to the development of a powerful new approach to study gene function (Dillon et al., 2005; Dykxhoorn et al., 2003; Elbashir et al., 2001; Hannon et al., 2004). Effective delivery of siRNA molecules into target cells or tissues is critical for successful RNA interference (RNAi) application. Here, we describe the use of human immunodeficiency virus type 1 (HIV‐1)–based lentiviral vectors for delivery of short hairpin RNA (shRNA), a precursor of siRNA, into primary neurons to suppress gene expression. Major advantages of lentiviral vectors are their ability to transduce nondividing cells and to confer long‐term expression of transgenes. This chapter covers selection of short hairpin sequences, vector design, production of lentiviral supernatants, transduction of dissociated primary hippocampal neurons, and testing the effectiveness of shRNA‐mediated silencing.

Introduction

Lentiviral vectors based on, for example, human immunodeficiency virus type 1 (HIV‐1) have been successfully used for effective gene silencing in neurons using RNAi (Cottrell 2004, Dittgen 2004). The lentiviral system bypasses deficiencies of other techniques such as transient transfections of siRNAs and the use of plasmid‐based mammalian expression vectors by allowing stable long‐lasting expression of shRNAs, which are processed in vivo to siRNAs, in cells that are difficult to transfect. Furthermore, in contrast to retroviral vectors derived from murine retroviruses, lentiviral vectors can efficiently infect both actively dividing, and nondividing postmitotic and terminally differentiated cells, such as brain and muscle cells (Kafri 1997, Naldini 1996, Van den Haute 2003). We and others have found lentiviral vectors to be valuable tools for stable gene silencing in a wide range of cell types, including neurons at different stages of development.

Section snippets

Lentiviral Vectors for shRNA Delivery

Most commonly used lentiviral vectors are derived from human immunodeficiency virus type 1 (HIV‐1). These vectors represent the most advanced lentiviral systems for gene delivery. Because they originate from viruses that are pathogenic for humans, the major emphasis in the construction of these vectors has been on their safety. The general strategy has been to use as few genetic elements of the original lentiviral genome as possible and to minimize the probability of generating a

Selection of siRNA Target Sequences and Design of shRNA Templates

Identification of optimal target sequences for siRNA is critical to achieve potent and specific gene silencing. The effectiveness of gene silencing depends on the sequence‐specific and thermodynamic properties of siRNA, as well as requires elaborate assessment of its specificity to minimize potential off‐target effects. Generally, shRNA contains a 19–29 nucleotide (nt) duplex connected by a short loop sequence (Siolas et al., 2005). Together they form a stem‐loop structure (“hairpin”) with a 3′

Production of Lentiviral Supernatants

Viral particles are produced by transient coexpression of the lentiviral vector along with either second‐ or third‐generation packaging constructs in human embryonic kidney (HEK) 293T cells, using the calcium phosphate coprecipitation method. We have been producing lentiviral particles using a second‐generation packaging system. The TRIP vector was pseudotyped with VSV‐G encoded by pMD.G (Dull et al., 1998). Gag, Pol, and Tat were expressed from packaging construct pCMVΔR8.91 (Zufferey et al.,

Lentiviral Transduction of Dissociated Hippocampal Neurons

Hippocampal neurons are typically prepared from rat fetuses at embryonic day 18–19 (E18‐E19) or from mouse fetuses at a comparable developmental stage. A number of protocols have been developed to isolate and culture hippocampal neurons (Banker et al., 1998). In the protocol we routinely use (see following), rat hippocampi are dissociated with trypsin. The dissociated cells are then plated at a medium density on poly‐l‐lysine or poly‐l‐lysine and laminin‐treated substrates and maintained in

Effectiveness and Specificity of shRNA‐Mediated Silencing

It is necessary to test how effectively each hairpin reduces the level of expression of the target gene. The degree of silencing should be verified at protein and/or mRNA level. Reduction in protein levels can be assessed by Western blot analysis, immunohistochemical analysis, and/or immunofluorescence‐based assays. Reduction of target mRNA levels can be evaluated by Northern blotting, RT‐PCR, QT‐PCR, and/or branched DNA (bDNA) assays (Gruber et al., 2005).

It is also imperative to ensure that

Acknowledgments

We thank Bruno Verhasselt (Ghent University, Belgium) for providing the TRIPΔU3‐EF1α‐EGFP and pSUPER(EcoRI) vectors. We also thank members of Van Aelst's and Skowronski's laboratories for sharing their expertise and helpful discussions. Our research is supported by NIH grants to L. V. A. (CA096882, CA64593) and J. S. (AI‐42561).

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