Document Detail


Apoptosis induced by DNA uptake limits transfection efficiency.
MedLine Citation:
PMID:  10585278     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
L H Li; A Sen; S P Murphy; G P Jahreis; H Fuji; S W Hui
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Experimental cell research     Volume:  253     ISSN:  0014-4827     ISO Abbreviation:  Exp. Cell Res.     Publication Date:  1999 Dec 
Date Detail:
Created Date:  2000-01-11     Completed Date:  2000-01-11     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0373226     Medline TA:  Exp Cell Res     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  541-50     Citation Subset:  IM    
Copyright Information:
Copyright 1999 Academic Press.
Affiliation:
Department of Cellular & Molecular Biophysics, Department of Immunology, Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo, New York 14263, USA.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Chloromethyl Ketones / pharmacology
Animals
Apoptosis / drug effects*,  physiology*
Caspases / antagonists & inhibitors
Cloning, Molecular / methods
Cysteine Proteinase Inhibitors / pharmacology
Electroporation / methods*
Genes, Reporter
Humans
K562 Cells / cytology,  enzymology
Luciferases / genetics
Mammary Neoplasms, Experimental
Mice
Plasmids / pharmacokinetics*
Transfection / methods*
Transgenes / physiology
Grant Support
ID/Acronym/Agency:
GM30969/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Amino Acid Chloromethyl Ketones; 0/Cysteine Proteinase Inhibitors; 0/butyloxycarbonyl-O-methyl-aspartyl-fluoromethyl ketone; EC 1.13.12.-/Luciferases; EC 3.4.22.-/Caspases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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