The use of specific AAV serotypes to stably transduce primary CNS neuron cultures.

Although primary neuronal cell cultures are a valuable source of in vitro insight for many neurobiologists, all current gene expression technologies for these cells have significant drawbacks. Some of these limitations of current gene expression protocols include toxicity, transient expression, a requirement for postnatal neurons, and/or low efficiency. To date, many types of experiments were not possible because of these limitations. Here, we outline a methodology by which primary cultured neurons can be transduced at any age, after plating, with virtually no toxicity and continued gene expression for the lifetime of the culture. This method involves the use of adeno-associated viral vectors, which have the potential to be highly useful for either upregulation or downregulation of single or multiple genes, including neurotrophins, other neuroprotective genes, and neurotoxins.

[1]  M. Dichter,et al.  Specific AAV serotypes stably transduce primary hippocampal and cortical cultures with high efficiency and low toxicity , 2008, Brain Research.

[2]  H. Paulson,et al.  RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia , 2004, Nature Medicine.

[3]  S. Kügler,et al.  Differential transgene expression in brain cells in vivo and in vitro from AAV-2 vectors with small transcriptional control units. , 2003, Virology.

[4]  J. Grieger,et al.  Packaging Capacity of Adeno-Associated Virus Serotypes: Impact of Larger Genomes on Infectivity and Postentry Steps , 2005, Journal of Virology.

[5]  P. Fan,et al.  Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. , 1996, Human gene therapy.

[6]  S. Kügler,et al.  Promoters and serotypes: targeting of adeno‐associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivo , 2005, Experimental physiology.

[7]  Yun Wang,et al.  Tropism and toxicity of adeno-associated viral vector serotypes 1, 2, 5, 6, 7, 8, and 9 in rat neurons and glia in vitro. , 2008, Virology.

[8]  J. Wolfe,et al.  Transduction characteristics of adeno-associated virus vectors expressing cap serotypes 7, 8, 9, and Rh10 in the mouse brain. , 2006, Molecular therapy : the journal of the American Society of Gene Therapy.

[9]  Zhijian Wu,et al.  Effect of genome size on AAV vector packaging. , 2010, Molecular therapy : the journal of the American Society of Gene Therapy.

[10]  R. Klein,et al.  AAV8, 9, Rh10, Rh43 vector gene transfer in the rat brain: effects of serotype, promoter and purification method. , 2008, Molecular therapy : the journal of the American Society of Gene Therapy.

[11]  D. Duan,et al.  Trans-splicing vectors expand the utility of adeno-associated virus for gene therapy. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[12]  M. Dragunow,et al.  Quantitative comparison of expression with adeno-associated virus (AAV-2) brain-specific gene cassettes , 2001, Gene Therapy.

[13]  M. Dichter,et al.  Calcium-dependent Paired-pulse Facilitation of Miniature Epsc Frequency Accompanies Depression of Epscs at Hippocampal Synapses in Culture , 1996 .

[14]  D. McCarty,et al.  Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis , 2001, Gene Therapy.

[15]  J. Mallet,et al.  Optimization of transgene expression at the posttranscriptional level in neural cells: implications for gene therapy. , 2003, Molecular therapy : the journal of the American Society of Gene Therapy.

[16]  Shu Wang,et al.  Transcriptional targeting to brain cells: Engineering cell type-specific promoter containing cassettes for enhanced transgene expression. , 2009, Advanced drug delivery reviews.

[17]  S. Kügler,et al.  Long-term in vivo and in vitro AAV-2-mediated RNA interference in rat retinal ganglion cells and cultured primary neurons. , 2005, Biochemical and biophysical research communications.