Adenovirus-mediated gene transfer into dissociated and explant cultures of rat hippocampal neurons.
Genetic manipulation offers great potential for studying the molecular and cellular processes which control or regulate the complex developmental properties of neurons. Gene transfer into neurons, however, is notoriously difficult. In this study we have used a replication-defective adenovirus (Adv/RSV beta gal), expressing beta-galactosidase (beta-gal) as a reporter gene, to infect dissociated cultures of rat hippocampal neurons and hippocampal slice cultures. Because future studies will require either long-term (e.g., developmental) or short-term (e.g., electrophysiological) expression of recombinant genes in neuronal cultures, we have optimized infection conditions for each situation. The Adv/RSV beta gal construct infects neurons and glial cells equally well, with no apparent alterations in cellular morphology. In slice cultures, the same efficiency and temporal control of beta-gal expression following Adv/RSV beta gal infection was achieved. Focal application of the adenoviruses, by microinjection, permitted infection of discrete subregions within the hippocampal explants. Whole cell recordings of dissociated hippocampal neurons and field recordings from the explant cultures, infected with Adv/RSV beta gal at low multiplicities of infection, indicated no significant alteration in the electrophysiological profiles of neurons in these cultures. The results demonstrate the utility of adenoviruses as gene transfer vectors for primary cultures of neurons. Adenovirus-mediated gene transfer into slice cultures also provides an opportunity to study development or plasticity in an environment where the circuitry and cytoarchitecture of the tissue are preserved and the areas of genetic manipulation can be spatially isolated.
Wilkemeyer
M F
MF
Department of Cell Biology, Baylor College of Medicine, Houston, Texas, USA.
Smith
K L
KL
Zarei
M M
MM
Benke
T A
TA
Swann
J W
JW
Angelides
K J
KJ
Eisensmith
R C
RC
D000256:Adenoviridae / Q000201:enzymology / Q000235:genetics*; D000818:Animals; D000831:Animals, Newborn; D002470:Cell Survival; D002478:Cells, Cultured; D004594:Electrophysiology; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism* / Q000502:physiology / Q000821:virology; D018408:Patch-Clamp Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D001616:beta-Galactosidase