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.
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.
Transduction and toxicity of AAV serotypes on cultured neurons at 2 weeks post-transduction
| Serotype | Dose (GC) | Percent Transduction (GFP+/DAPI+) | Total Cells Counted (n) | Cultures (n) | Percent of transduced cells that are neurons (MAP2+GFP+/GFP+) | Total Cells Counted (n) | Cultures (n) |
|---|---|---|---|---|---|---|---|
| Naturally occuring | |||||||
| 1 | 2.5 × 1011 | 94.1 ± 1.9% | 739 | 4 | 79.6 ± 4.1% | 295 | 3 |
| 2 | 2.0 – 2.5 × 1011 | 83.8 ± 4.0% | 736 | 5 | 53.6 ± 9.0% | 533 | 4 |
| 5 | 2.5 × 1011 | toxic | 2 | – | |||
| 2.5 × 109 | 48.4 ± 9.4% | 1074 | 3 | 61.9 ± 17.7% | 227 | 3 | |
| 6 | 2.5 × 1011 | toxic | 3 | – | |||
| 6.7 × 1010 | toxic | 6 | – | ||||
| 2.5 – 5.0 × 109 | 72.1 ± 19.1% | 822 | 3 | 84.7 ± 10.4% | 264 | 3 | |
| 7 | 2.5 × 1011 | 88.0 ± 4.3% | 785 | 5 | 74.4 ± 12.8% | 392 | 4 |
| 8 | 2.5 × 1011 | 94.2 ± 1.9% | 599 | 5 | 63.2 ± 13.9% | 412 | 4 |
| 9 | 2.5 × 1011 | 79.5 ± 5.8% | 985 | 4 | 43.2 ± 2.2% | 320 | 3 |
| Engineered variants | |||||||
| 2.hu29R | 3.1 × 1011 | 0% | 3 | – | |||
| 6.1 | 5.0 × 1011 | 91.3 ± 1.7% | 491 | 3 | 64.1 ± 4.1% | 399 | 3 |
| 6.2 | 5.0 × 1010 | toxic | 3 | – | |||
| 6.1.2 | 5.0 × 1011 | 94.1 ± 2.8% | 443 | 3 | 85.1 ± 2.5% | 282 | 3 |
All vectors listed in this table encoded CMV-GFP. Vectors were added to cells at 7DIV; counts were performed at 21DIV.
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.
Refer to Brain Res. 2008 Jan 23; 1190: 15–22.