High Ca(2+)-phosphate transfection efficiency enables single neuron gene analysis.
Introducing exogenous genes into cells is one of the most important molecular techniques to study gene functions. Comparing to other type of cells, neurons are more difficult to transfect with cDNAs because they are very sensitive to microenvironmental changes. Among various gene transfer techniques, the Ca(2+)-phosphate transfection method is one of the most popular tools in neuroscience research because of its low cell toxicity and easiness to use. However, it is well known that the Ca(2+)-phosphate transfection efficiency in neurons is very low, typically in the range of 1-5%, which has limited its applications in gene functional analyses. Here we report a novel Ca(2+)-phosphate transfection protocol that dramatically increased the transfection efficiency by 10-fold, up to 60%, while maintaining low cell toxicity. The critical factors are the formation of homogenous snow-like precipitate with particle size about 1-3 microm and the subsequent removal of the precipitate. Using this new transfection protocol, we were able to routinely transfect single autaptic neurons in hippocampal microisland cultures and combine it with electrophysiology and fluorescent imaging methods to study gene functions. This high efficiency, low toxicity, and simple to use gene transfer method will have a broad application in gene research at the single cell level.
Jiang
M
M
Department of Biology, The Pennsylvania State University, University Park, PA 16802, USA.
Deng
L
L
Chen
G
G
D000818:Animals; D000831:Animals, Newborn; D002130:Calcium Phosphates; D046508:Culture Techniques / Q000379:methods; D018076:DNA, Complementary; D006624:Hippocampus; D009474:Neurons / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection / Q000379:methods*
D002130:Calcium Phosphates; D018076:DNA, Complementary; C020243:calcium phosphate