Apoptosis induced by DNA uptake limits transfection efficiency.
Electrotransfection is an effective method for transfecting lymphoid cells. However, the transfection efficiency of certain lymphoid cells is low. L1210 subclones and NFS-70 pro-B cells, which are highly refractory to various transfection methods, were used to identify the limiting factors. Cells were electrotransfected with plasmids coding for green fluorescence protein or luciferase. The luciferase expression of L1210 subclone 3-3 was found to increase 6-12 h after electroporation, but decreased significantly from 12 to 48 h. The lower level of luciferase activity at later time periods correlated with decreases in cell viability, which was shown to be due to apoptosis, as determined by propidium iodide/acrindine orange staining, DNA laddering, and prevention of cell death by addition of caspase inhibitors. Similar results were observed with NFS-70 pro-B cells and select L1210 subclones. In contrast, L1210 parental and L1210 subclone 7-15.6 cells undergo only low levels of apoptosis (< or = 5%). Apoptosis occurred only when DNA (plasmids or salmon sperm DNA) was present during electroporation, but was not dependent on the conformation of the DNA used or the expression of transgenes. Cells pulsed in the presence of dextran sulfate (MW 500,000) did not apoptose. Similar results were observed when L1210 subclone 3-3 was transfected using the cationic lipid 1, 2-dioleoyl-3-trimethylammonium propane, although the transfection efficiency and corresponding rate of apoptosis were significantly lower. Applying the caspase inhibitor fluoromethyl ketone (Boc-ASP-FMK) dramatically improved cell viability and transgene expression of select L1210 subclones and NFS-70 pro-B cells.
Li
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LH
Department of Cellular & Molecular Biophysics, Department of Immunology, Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo, New York 14263, USA.
Sen
A
A
Murphy
S P
SP
Jahreis
G P
GP
Fuji
H
H
Hui
S W
SW
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology*; D061945:Caspase Inhibitors; D003001:Cloning, Molecular / Q000379:methods; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D018274:Electroporation / Q000379:methods*; D017930:Genes, Reporter; D006801:Humans; D020014:K562 Cells / Q000166:cytology / Q000201:enzymology; D008156:Luciferases / Q000235:genetics; D008325:Mammary Neoplasms, Experimental; D051379:Mice; D010957:Plasmids / Q000493:pharmacokinetics*; D014162:Transfection / Q000379:methods*; D019076:Transgenes / Q000502:physiology
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; C103192:butyloxycarbonyl-O-methyl-aspartyl-fluoromethyl ketone; D008156:Luciferases