Electroporation of primary neural cultures: a simple method for directed gene transfer in vitro.
Gene transfer into cells of the nervous system is an important method to analyze tissue-specific gene functions. Although highest transfection efficiencies are generally obtained by viral gene transfer, non-viral methods are attractive because they are less labor intensive and more suitable for high throughput screening approaches. Here we describe an approach for electroporation-based gene transfer into primary neural cells isolated from dissociated murine cerebella. Using GFP as reporter molecule, we show that electroporation allows for efficient gene transfer into embryonic and postnatal neural cells under highly controlled experimental conditions. Furthermore we show that adaptation of electroporation parameters allowed for the preferential transfection of subsets of neural cells within the mixed primary culture. Using electroporation settings of high voltage and low capacitance (500 V/50 microF) we achieved a transfection efficiency of about 10% of small neural cells which were identified as granule cells by the expression of the granule cell-specific marker NeuN. At electroporation settings of 220 V/975 microF, large and stellate-shaped cells that comprised about 10% of the GFAP-positive population of astrocytes were preferentially transfected. We conclude that electroporation of primary neural cells can be used to target gene transfer to subsets of neural cells.
Mertz
Kirsten D
KD
Anatomisches Institut, Anatomie und Zellbiologie, Rheinische Freidrich-Wilhelms-Universität Bonn, Germany.
Weisheit
Gunnar
G
Schilling
Karl
K
Lüers
Georg H
GH
D000818:Animals; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000254:growth & development; D018274:Electroporation / Q000379:methods*; D005260:Female; D005333:Fetus; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection / Q000379:methods*
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins