Favre, D. and Trépo, C.

Translational extracts active biologically in vitro obtained from eukaryotic monolayer cells: a versatile method for viral RNA studies.

J. Virol. Methods 92 (2):177-181 (2001). 

Protein synthesis is the cellular phenomenon by which ribosomes are translating the messenger RNA into protein (reviewed in Translational control, Cold Spring Harbour Laboratory Press, 1996). The following figure does summarize the whole process in eukaryotic cells in vivo:

Several in vitro protein-synthesizing systems have been used in recent years for the translation of viral, prokaryotic, as well as eukaryotic messenger ribonucleic acids (mRNAs). Of these, the rabbit reticulocyte lysate, the Krebs ascites fluid (both of animal origin), and the wheat germ extract have received the most attention. However, these systems are not representative of eukaryotic cells in the way they regulate translation. They cannot or do not respond to various physiological (eg: hormones, toxins, ions), chemical, and other external (heat shock, magnetic fields) stimuli which are important regulators of cellular functions in nucleated cells, and they cannot be used for studies on viral infection. In addition to these widely used systems, cell-free extracts originating from a variety of eukaryotic cell types have been generated for the translation of mRNAs or to study aspects of the regulation of protein synthesis.

The generation of an efficient protein-synthesizing system obtained from eukaryotic cells that have grown as monolayers has been hampered for a long time by the lack of translatability of exogenously added mRNAs, and by the loss of biological activities after freeze/thawing.

Indeed, the successful generation of a translational extract obtained from eukaryotic cells that have grown as monolayers has an important potential:

i)                                  it can be employed in place of the mostly used commercially available rabbit reticulocyte lysate;

ii)                                it is a priori preferable, since no living animals are required for its generation through experimental chronic anemia;

iii)                              the cells grown as monolayers in small volumes of cell culture medium, and not as large scale suspension cultures, can be preincubated with hormones, toxins, ions, thus allowing modulation of biological processes prior to extract preparation;

iv)                              the cells can be infected with viruses prior to extract preparation;

v)                                the translation extract allows both cap-dependent and cap-independent translation;

vi)                              in the presence of sucrose, it allows freeze/thawing without loss of activity, here the translatability of exogenously added mRNAs, a features that can be extended to various biological samples for the preservation of their respective enzymatic activities.

vii)                            It can lead to the study of the fate of the HCV IRES in vitro.