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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Ejeskär, Katarina,1969Gothenburg University,Göteborgs universitet,Institutionen för biomedicin, avdelningen för medicinsk genetik och klinisk genetik,Institute of Biomedicine, Department of Medical and Clinical Genetics (författare)

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.

Artikel/kapitelEngelska2006

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2006

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LIBRIS-ID:oai:gup.ub.gu.se/54862
https://gup.ub.gu.se/publication/54862URI

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Språk:engelska

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Ämneskategori:ref swepub-contenttype
Ämneskategori:art swepub-publicationtype

Anmärkningar

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.

Ämnesord och genrebeteckningar

Amino Acid Sequence
Cell Line
Tumor
Cell Nucleus
chemistry
Cell Proliferation
Cytoplasm
chemistry
Green Fluorescent Proteins
analysis
genetics
toxicity
Humans
Molecular Sequence Data
Nuclear Localization Signals
genetics
RNA
Messenger
biosynthesis
genetics
Transfection
methods
Trypsin
pharmacology

Biuppslag (personer, institutioner, konferenser, titlar ...)

Fransson, Susanne,1975Gothenburg University,Göteborgs universitet,Institutionen för biomedicin, avdelningen för medicinsk genetik och klinisk genetik,Institute of Biomedicine, Department of Medical and Clinical Genetics(Swepub:gu)xfrans (författare)
Zaibak, Faten (författare)
Ioannou, Panayiotis A (författare)
Göteborgs universitetInstitutionen för biomedicin, avdelningen för medicinsk genetik och klinisk genetik (creator_code:org_t)

Sammanhörande titlar

Ingår i:International journal of molecular medicine17:6, s. 1011-61107-3756

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https://gup.ub.gu.se/publication/54862

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International journal of molecular medicine (Sök värdpublikationen i LIBRIS)

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Av författaren/redakt...: Ejeskär, Katarin ...; Fransson, Susann ...; Zaibak, Faten; Ioannou, Panayio ...

Artiklar i publikationen: International jo ...

Av lärosätet: Göteborgs universitet

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