Document Detail

Method for efficient transfection of in vitro-transcribed mRNA into SK-N-AS and HEK293 cells: difference in the toxicity of nuclear EGFP compared to cytoplasmic EGFP.
MedLine Citation:
PMID:  16685409     Owner:  NLM     Status:  MEDLINE    
Here we report a method for efficient transfection of in vitro-transcribed mRNA into two different types of human adherent cells, the neuroblastoma cell line SK-N-AS, and the transformed kidney cell line HEK293. By using newly trypsinized adherent cells in suspension and Lipofectaminetrade mark 2000, we detected a transfection efficiency of 80-90% in both cell lines and a cell viability of 90% in SK-N-AS and 60% in HEK293, 24 h after transfection when using cytoplasmic enhanced green fluorescent protein (EGFP)-mRNA. We have evaluated the different effects of the generally used EGFP that mainly localizes to the cytoplasm and nuclear EGFP, where the nuclear EGFP are more toxic to the cells than the cytoplasmic EGFP. In order to develop a null experiment, we constructed a short non-functional mRNA including a nuclear localization signal and evaluated the concentrations at which mRNA encoding nuclear proteins can be added without a general toxicity, depending on the fact that the proteins are localized to the nucleus. For both SK-N-AS and HEK293 cells, a concentration of up to 100 ng mRNA in 10(5) cells, encoding a nuclear protein with no other function, did not affect the cells. For evaluation of the method, we screened four different human mRNAs, PDG, DFFA, CORT and PEX14, for their ability to affect cell proliferation in these cells. PEX14 was the only gene that significantly (p=0.03) reduced cell proliferation for both cell types, DFFA significantly (p=0.04) reduced cell proliferation in SK-N-AS but not in HEK293 cells. PGD and CORT did not have any effect on cell proliferation. We have developed an easy method for efficient delivery of in vitro-transcribed mRNA into the adherent cell lines, SK-N-AS and HEK293. This method is useful for a quick screening of how different genes affect cell proliferation.
Katarina Ejeskär; Susanne Fransson; Faten Zaibak; Panayiotis A Ioannou
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  International journal of molecular medicine     Volume:  17     ISSN:  1107-3756     ISO Abbreviation:  Int. J. Mol. Med.     Publication Date:  2006 Jun 
Date Detail:
Created Date:  2006-05-10     Completed Date:  2006-07-10     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  9810955     Medline TA:  Int J Mol Med     Country:  Greece    
Other Details:
Languages:  eng     Pagination:  1011-6     Citation Subset:  IM    
Murdoch Children's Research Institute, Melbourne University, Royal Children's Hospital, Australia.
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MeSH Terms
Amino Acid Sequence
Cell Line, Tumor
Cell Nucleus / chemistry*
Cell Proliferation
Cytoplasm / chemistry*
Green Fluorescent Proteins / analysis*,  genetics,  toxicity*
Molecular Sequence Data
Nuclear Localization Signals / genetics
RNA, Messenger / biosynthesis,  genetics*
Transfection / methods*
Trypsin / pharmacology
Reg. No./Substance:
0/Nuclear Localization Signals; 0/RNA, Messenger; 0/enhanced green fluorescent protein; 147336-22-9/Green Fluorescent Proteins; EC

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