A method of mRNA production for use in
transfection is provided, that involves
in vitro transcription of PCR generated templates with specially designed primers, followed by polyA addition, to produce a construct containing 3′ and 5′ untranslated sequence (“UTR”), a 5′ cap and / or
Internal Ribosome Entry Site (IRES), the
gene to be expressed, and a polyA
tail, typically 50-2000 bases in length. This
RNA can efficiently transfect different kinds of cells. This approach results in increased efficiency (fidelity and productivity) of mRNA synthesis and is less
time consuming because it does not require
cloning, and also consequently eliminates the unwanted errors and effects related to
RNA made on
DNA templates obtained with
cloning techniques. The results of
transfection of RNAs demonstrate that
RNA transfection can be very effective in cells that are exceedingly difficult to transfect efficiently with
DNA constructs. Further, the levels of
gene expression following
mRNA transfection are consistent from
cell to
cell in an experiment and these levels can be controlled over a wide range simply by changing the amount of mRNA that is transfected, and without obvious cytotoxic effects due to the levels of RNA per se. Due to high efficiency the cells can be simultaneously transfected with multiple genetic constructs. The method can be used to deliver genes into cells not- or only poorly transfectable for
DNA,
in vitro and
in vivo.