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Comparative Study
. 2008 Jan 23:1190:15-22.
doi: 10.1016/j.brainres.2007.11.015. Epub 2007 Nov 17.

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

Nicolas C Royo  1 , Luk H Vandenberghe, Jing-Yuan Ma, Alisse Hauspurg, Liya Yu, Margaret Maronski, Julie Johnston, Marc A Dichter, James M Wilson, Deborah J Watson
Affiliations
Comparative Study

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

Nicolas C Royo et al. Brain Res. .

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.

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Figures

Figure 1
Figure 1
Transduction of cultured neurons and astrocytes by AAV serotypes. (A-B) Hippocampal cultures transduced by AAV2 at 4 weeks in vitro and harvested at 5 weeks in vitro. Green is intrinsic GFP fluorescence. (C) Hippocampal cultures transduced by AAV2 at 4 weeks in vitro and harvested at 8 weeks in vitro. Green is intrinsic GFP fluorescence; red is MAP2 immunostaining for neurons. (D-G) Cortical cultures transduced at 4 weeks in vitro and harvested at 5 weeks in vitro. Green is intrinsic GFP fluorescence. (D) AAV1 transduction of neurons. (E) AAV7 transduction of neurons (left) and astrocytes (right). (F) AAV9 transduction of neurons. (G) AAV9 transduction of astrocytes. Bar in A, C-G, 50 microns. Bar in B, 5 microns.
Figure 2
Figure 2
Expression of GFP in neurons and astrocytes of hippocampal cultures transduced by AAV1 (A-F), AAV7 (G-L), or AAV9 (M-R). GFP expression is shown in the first column (A, D, G, J, M R) and GFAP immunostaining (B, H, N) or MAP2 immunostaining (E, K, Q) in the second column. The third column contains a merged pseudocolored representation of the first two panels in each row. Bar, 100 microns.
Figure 3
Figure 3
Glutamate toxicity and NT-4/5 protection in AAV1-transduced primary hippocampal cultures in a 96-well plate. Cultures were transduced at DIV7 with AAV1-CMV-GFP. (A) Increasing concentrations of L-glutamate (0, 0.3, 1.9, 11.1, 66.6 or 400 microM) were added to wells (n=3 per dose) at DIV14 for 24 hr. Values are normalized to the untreated well (100%). Inset, monochrome fluorescent photomicrograph of transduced (GFP expressing) cells in a 96-well plate. (B) Cultures were treated with increasing concentrations of NT-4/5 (0, 10, 33, 100 or 300 ng/ml) for 24 hr before exposure to 3.7 mM (filled circles) or 11.1 mM (filled squares) of L-glutamate for 24 hr.
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References

    1. Asokan A, Hamra JB, Govindasamy L, Agbandje-McKenna M, Samulski RJ. Adeno-associated virus type 2 contains an integrin alpha5beta1 binding domain essential for viral cell entry. J Virol. 2006;80:8961–8969. - PMC - PubMed
    1. Broekman ML, Comer LA, Hyman BT, Sena-Esteves M. Adeno-associated virus vectors serotyped with AAV8 capsid are more efficient than AAV-1 or -2 serotypes for widespread gene delivery to the neonatal mouse brain. Neurosci. 2006;138:501–510. - PubMed
    1. Burger C, Gorbatyuk OS, Velardo MJ, Peden CS, Williams P, Zolotukhin S, Reier PJ, Mandel RJ, Muzyczka N. Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system. Mol Ther. 2004;10:302–317. - PubMed
    1. Carrier RL, Ma TC, Obrietan K, Hoyt KR. A sensitive and selective assay of neuronal degeneration in cell culture. J Neurosci Meth. 2006;154:239–244. - PubMed
    1. Cearley C, Wolfe JH. Transduction characteristics of adeno-associated virus vectors expressing cap serotypes 7, 8, 9 and Rh10 in the mouse brain. Mol Ther. 2006;13:528–537. - PubMed

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