Elsevier

Brain Research

Volume 1190, 23 January 2008, Pages 15-22
Brain Research

Research Report
Specific AAV serotypes stably transduce primary hippocampal and cortical cultures with high efficiency and low toxicity

https://doi.org/10.1016/j.brainres.2007.11.015Get rights and content

Abstract

Most current methods of gene delivery for primary cultured hippocampal neurons are limited by toxicity, transient expression, the use of immature neurons and/or low efficiency. We performed a direct comparison of seven serotypes of adeno-associated virus (AAV) vectors for genetic manipulation of primary cultured neurons in vitro. Serotypes 1, 2, 7, 8 and 9 mediated highly efficient, nontoxic, stable long-term gene expression in cultured cortical and hippocampal neurons aged 0–4 weeks in vitro; serotypes 5 and 6 were associated with toxicity at high doses. AAV1 transduced over 90% of all cells with approximately 80% of the transduced cells being neurons. The method was readily adapted to a high-throughput format to demonstrate neurotrophin-mediated neuroprotection from glutamate toxicity in cultured neurons at 2 weeks in vitro. These vectors should prove highly useful for efficient overexpression or downregulation of genes in primary neuronal cultures at any developmental stage.

Introduction

Cultures of dissociated primary neurons and astrocytes are frequently used by neurobiologists to study neuronal physiology and pathophysiology. A variety of methods have been used for gene delivery into cultured neurons, including recombinant Sindbis, SV40 or Semliki-forest viral vectors as well as plasmid transfection using calcium phosphate, commercially available lipids or electroporation. However, each of these methods is limited by one or more of the following drawbacks: very low efficiency, short-term gene expression, toxicity, inability to express common reporter genes and/or the requirement to transfect immature neurons (reviewed in Craig, 1998). Although transient, low-efficiency gene expression is sufficient for some types of experiments, biochemical experiments require much higher efficiency.

Adeno-associated viral (AAV) vectors readily transduce neurons in vivo with low toxicity. Here, we report in vitro transduction and toxicity patterns for AAV vectors with 7 different serotypes and 4 AAV vectors with engineered capsids. Several of these vectors mediated efficient, stable and nontoxic transduction of hippocampal and cortical neurons in vitro.

Section snippets

Tropism and efficiency

Recombinant AAV vectors were generated using a CMV-GFP expression cassette as the genome and packaged using capsid sequences from AAV1, 2, 5, 6, 7, 8 or 9. To compare transduction efficiencies of the different AAV serotypes, a single dose of 2.0 − 2.5 × 1011 genome copies (GC) of each vector was added to cultured rat hippocampal cells on day in vitro (DIV) 7. By DIV21, the cultures exposed to serotypes 1, 2, 7, 8 and 9 showed remarkable levels of transduction, approximately 80–94% of the cells in

Discussion

Current methods for gene transfer into cultured neurons are of limited utility due to low efficiency, high toxicity, short duration of gene expression, or the restriction that gene delivery must be performed in immature cells on the day of plating. Transgenic animals can be a source of genetically modified cultures, but generation of these animals is expensive, labor-intensive and mostly restricted to mice. Our results demonstrate that the use of AAV vectors can overcome all of these problems.

Neuronal cultures

The standard culture technique was described previously (Cummings et al., 1996). Briefly, cortices or hippocampi from E19 Sprague–Dawley rat embryos were trypsinized in Dulbecco's minimum essential medium (DMEM; Whittaker Bioproducts) containing 0.027% trypsin at 4 °C for 20 min. They were triturated in media consisting of DMEM supplemented with 10% bovine calf serum (Hyclone), 10% Ham's F12 with glutamine (Whittaker Bioproducts) and 50 U/ml penicillin–streptomycin (Sigma). Dissociated cells

Acknowledgments

This research was supported by grants from the National Institutes of Health RO1 NS040978 (DJW), P30 DK 47757-09 and P01 HL 59407-03 (JMW) and RO1 NS24260 (MAD), by a pilot grant from the University of Pennsylvania Institute for Medicine and Engineering (DJW) and by startup funds from the University of Pennsylvania Department of Neurosurgery (DJW). We thank the Vector and Quality Control Cores of the University of Pennsylvania for producing the vectors, Dr. Virginia Lee for the MAP2 antibody,

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