Toxic effects of lipid-mediated gene transfer in ventral mesencephalic explant cultures.
Adverse effects of cDNA and oligonucleotide delivery methods have not yet been systematically analyzed. We introduce a protocol to monitor toxic effects of two non-viral lipid-based gene delivery protocols using CNS primary tissue. Cell membrane damage was monitored by quantifying cellular uptake of propidium iodide and release of cytosolic lactate dehydrogenase to the culture medium. Using a liposomal transfection reagent, cell membrane damage was already seen 24 hr after transfection. Nestin-positive target cells, which were used as morphological correlate, were severely diminished in some areas of the cultures after liposomal transfection. In contrast, the non-liposomal transfection reagent revealed no signs of toxicity. This approach provides easily accessible information of transfection-associated toxicity and appears suitable for prescreening of transfection reagents.
Bauer
Matthias
M
GSF-National Research Center for Environment and Health, Institute for Human Genetics, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany.
Kristensen
Bjarne Winther
BW
Meyer
Morten
M
Gasser
Thomas
T
Widmer
Hans Ruedi
HR
Zimmer
Jens
J
Ueffing
Marius
M
D000818:Animals; D002462:Cell Membrane / Q000187:drug effects; D004247:DNA / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007202:Indicators and Reagents / Q000633:toxicity; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008055:Lipids / Q000633:toxicity*; D008081:Liposomes / Q000633:toxicity*; D008636:Mesencephalon / Q000187:drug effects* / Q000196:embryology / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011419:Propidium / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection / Q000379:methods*
C498175:Effectene; D007202:Indicators and Reagents; D008055:Lipids; C086724:Lipofectamine; D008081:Liposomes; D049452:Green Fluorescent Proteins; D011419:Propidium; D004247:DNA; D007770:L-Lactate Dehydrogenase