Calcium phosphate-mediated transfection of primary cultured brain neurons using GFP expression as a marker: application for single neuron electrophysiology.
We investigated the efficiency of transfecting primary cultured rat postnatal brain neurons (substantia nigra pars compacta neurons and locus coeruleus neurons) with cDNA encoding GFP (jellyfish green fluorescent protein) using a calcium phosphate method. The proportion of transfected neurons (transfection efficiency) was approximately 5%, when cultures from the substantia nigra pars compacta were transfected 3 days after plating. The transfection efficiency decreased when cultures were transfected 10 days after plating (1.7%). Neurons were cotransfected at a very high probability ( > 78%) with the muscarinic m2-receptor cDNAs together with GFP plasmids. Transfected neurons were very healthy as indicated by the zero-current potential and the microscopical appearance. Because the transfection efficiency is low, this method cannot be used for experiments involving the whole cell population. The transfection efficiency of 1.7% corresponded to approximately 20 transfected cells per dish in our culture conditions and these cells are sufficient in number for electrophysiological studies. Therefore, this is an excellent method for studying the influence of exogenous genes on single neurons using electrophysiological techniques.
Watanabe
S Y
SY
Department of Anatomy and Cell Biology, University of Illinois at Chicago, College of Medicine, 60612, USA.
Albsoul-Younes
A M
AM
Kawano
T
T
Itoh
H
H
Kaziro
Y
Y
Nakajima
S
S
Nakajima
Y
Y
D000818:Animals; D002130:Calcium Phosphates / Q000494:pharmacology*; D002478:Cells, Cultured; D004594:Electrophysiology; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism*; D008125:Locus Coeruleus / Q000166:cytology / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism* / Q000502:physiology; D051381:Rats; D020318:Rats, Long-Evans; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism; D013378:Substantia Nigra / Q000166:cytology / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D002130:Calcium Phosphates; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011976:Receptors, Muscarinic; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; D049452:Green Fluorescent Proteins; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; C485829:calcium phosphate, dibasic, anhydrous