Specific AAV serotypes stably transduce primary hippocampal and cortical cultures with high efficiency and low toxicity.
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.
Department of Neurosurgery, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Nicolas C
NC
Royo
Luk H
LH
Vandenberghe
Jing-Yuan
JY
Ma
Alisse
A
Hauspurg
Liya
L
Yu
Margaret
M
Maronski
Julie
J
Johnston
Marc A
MA
Dichter
James M
JM
Wilson
Deborah J
DJ
Watson
D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism / Q000821:virology; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D000229:Dependovirus / Q000145:classification* / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology; D009474:Neurons / Q000166:cytology / Q000378:metabolism* / Q000821:virology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012703:Serotyping; D014161:Transduction, Genetic / Q000379:methods*; D014162:Transfection / Q000379:methods*; D019076:Transgenes / Q000235:genetics*
D049452:Green Fluorescent Proteins