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.

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Fältnamn	Indikatorer	Metadata
000		03985naa a2200541 4500
001		oai:gup.ub.gu.se/54862
003		SwePub
008		240910s2006 \| \|\|\|\|\|\|\|\|\|\|\|000 \|\|eng\|
024	7	a https://gup.ub.gu.se/publication/548622 URI
040		a (SwePub)gu
041		a eng
042		9 SwePub
072	7	a ref2 swepub-contenttype
072	7	a art2 swepub-publicationtype
100	1	a Ejeskär, Katarina,d 1969u Gothenburg University,Göteborgs universitet,Institutionen för biomedicin, avdelningen för medicinsk genetik och klinisk genetik,Institute of Biomedicine, Department of Medical and Clinical Genetics4 aut0 (Swepub:gu)xejeka
245	1 0	a 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.
264	1	c 2006
520		a 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.
653		a Amino Acid Sequence
653		a Cell Line
653		a Tumor
653		a Cell Nucleus
653		a chemistry
653		a Cell Proliferation
653		a Cytoplasm
653		a chemistry
653		a Green Fluorescent Proteins
653		a analysis
653		a genetics
653		a toxicity
653		a Humans
653		a Molecular Sequence Data
653		a Nuclear Localization Signals
653		a genetics
653		a RNA
653		a Messenger
653		a biosynthesis
653		a genetics
653		a Transfection
653		a methods
653		a Trypsin
653		a pharmacology
700	1	a Fransson, Susanne,d 1975u Gothenburg University,Göteborgs universitet,Institutionen för biomedicin, avdelningen för medicinsk genetik och klinisk genetik,Institute of Biomedicine, Department of Medical and Clinical Genetics4 aut0 (Swepub:gu)xfrans
700	1	a Zaibak, Faten4 aut
700	1	a Ioannou, Panayiotis A4 aut
710	2	a Göteborgs universitetb Institutionen för biomedicin, avdelningen för medicinsk genetik och klinisk genetik4 org
773	0	t International journal of molecular medicineg 17:6, s. 1011-6q 17:6<1011-6x 1107-3756
856	4 8	u https://gup.ub.gu.se/publication/54862

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