Generation of a translational extract from eukaryotic cells that have grown as monolayers

Abstract

The present article relates to the rapid and efficient generation of an in vitro translational extract that is obtained from the cytoplasmic fraction of eukaryotic cells that have grown as monolayers. The procedure is totally devoid of the use of animals or animal fluids. The cytoplasmic extract that is obtained allows efficient protein synthesis of exogenously added mRNAs. The latter mRNAs can be purified from prokaryotic or eukaryotic cells, or can be transcribed in vitro by employing any convenient RNA polymerase (for example, the bacteriophage SP6, T3 or T7 RNA polymerase). The described cytoplasmic preparation appears to be applicable to a large number of different eukaryotic cell lines. The cytoplasmic extract can be prepared freshly on a daily basis, or can be frozen and subsequently thawed for further use, without loss of activity. The preparation of the translation extract does not require living animals.