Chemically controlled formation of a DNA/calcium phosphate coprecipitate: application for transfection of mature hippocampal neurons.
Numerous methods exist for transfecting postmitotic neurons, for example, DNA/calcium phosphate coprecipitation, cationic lipids, viruses, and physical methods such as microinjection, electroporation, and biolistics. Most methods, however, are either toxic to the cell, yield only poor transfection efficiencies, or cells have to be electroporated before plating. In this article, we present a standardized and fast transfection method using DNA/calcium phosphate coprecipitates that efficiently transfer DNA into mature, postmitotic hippocampal neurons. Shifting to CO(2)-independent media with a well-defined pH allows for the tight control of the coprecipitate formation and for adjusting the transfection parameters for the individual DNA plasmid used. The two critical parameters for reproducible and efficient transfections are: the precise pH during crystal formation, and the incubation time of the cells with the coprecipitate. This improved procedure now enables biochemical approaches. By transfecting a dominant-positive Ras mutant, we activate the Erk/MAP kinase signal transduction pathway. Furthermore, using a siRNA plasmid directed against MAP2, the level of an endogenously expressed protein is down-regulated upon transfection. These two approaches demonstrate that the presented transient transfection method can now be used to address questions on a biochemical level in hippocampal neurons.
Goetze
Bernhard
B
Max-Planck-Institute for Developmental Biology, Tübingen, Germany.
Grunewald
Barbara
B
Baldassa
Simona
S
Kiebler
Michael
M
D000818:Animals; D002130:Calcium Phosphates / Q000138:chemical synthesis* / Q000494:pharmacology*; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology; D004247:DNA / Q000138:chemical synthesis* / Q000235:genetics / Q000494:pharmacology*; D003712:Dendrites / Q000187:drug effects / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D048049:Extracellular Signal-Regulated MAP Kinases / Q000187:drug effects / Q000378:metabolism; D005333:Fetus; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D018715:Microscopy, Video; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D010957:Plasmids / Q000187:drug effects / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D013997:Time Factors; D014162:Transfection / Q000379:methods*; D018631:ras Proteins / Q000235:genetics
D002130:Calcium Phosphates; D003470:Culture Media; C489787:MAP2 protein, rat; D008869:Microtubule-Associated Proteins; D034741:RNA, Small Interfering; C055197:calcium phosphate-DNA precipitate; D004247:DNA; D048049:Extracellular Signal-Regulated MAP Kinases; D018631:ras Proteins