High throughput screening method for identification of new lipofection reagents.
Lipofection, the transfer of genetic material into cells by means of cationic lipids, is of growing interest for in vitro and in vivo approaches. In order to identify ideal lipofection reagents in a HTS, we have developed an automated lipofection method for the transfer of reporter genes into cells and for determination of the lipofection results. The method has specifically been designed and optimized for 96-well microtiter plates and can successfully be carried out by a pipetting robot with accessory equipment. It consists of two separate parts: (1) pretransfection (preparation of liposomes, formation of lipoplexes, and lipoplex transfer to the cells) and (2) posttransfection (determination of the reporter enzyme activity and the protein content of the transfected cells). Individual steps of the lipofection method were specifically optimized - for example, lipoplex formation and incubation time as well as cell lysis, cell cultivating, and the reporter gene assay. The HTS method facilitates characterization of the transfection properties (efficiency and cytotoxicity) of large numbers of (cationic) lipids in various adherent cell types.
Regelin
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AE
Tumor Biology Center, Department of Clinical Research/Phospholipids, Freiburg, Germany.
Fernholz
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E
Krug
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HF
Massing
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U
D000818:Animals; D001331:Automation; D019556:COS Cells; D002412:Cations / Q000378:metabolism*; D002448:Cell Adhesion; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation* / Q000379:methods*; D006801:Humans; D050356:Lipid Metabolism; D008081:Liposomes / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
D002412:Cations; D008081:Liposomes; D004247:DNA