Site Directed Mutagenesis in Ifnβ Gene in Order to Increasing Its mRNA Stability and Translation Level Using Megaprimer PCR Method

Beta interferon (IFNβ) protein is produced as a recombinant drug and used in treatment of some diseases like Multiple Sclerosis. In eukaryotic cells, IFNβ mRNA is rapidly degraded and its half-life is too short. One of the contributing factors to this short half-life is presence of the AU rich element (ARE) in 3´UTR of this mRNA. This region has an inhibitory effect on translation too. Our aim in this research was to delete ARE from IFNβ gene in order to increase its mRNA stability and translational level.
In order to delete an 18 nucleotide sequence from ARE, the Megaprimer PCR technique was used. The PCR product was digested with EcoRI and BglII enzymes. The vector was partially digested with the same enzymes. The digested PCR product was purified and cloned into the vector. Then, the recombinant vectors were transfected into CHO cell line.
The first PCR reaction product contained a deletion mutation and was used as megaprimer in the second reaction. Partial digestion of the vector yielded a variety of fragments with different weights. The sufficient fragment was purified from the gel and used as a cloning vector. Final product of PCR was cloned into the vector. The accuracy of the cloning reaction was confirmed and the recombinant vector was transfected into CHO cell line.
An 18 nucleotide region of IFNβ mRNA was deleted. The influence of this microdeletion on mRNA stability and translational efficiency needs to be surveyed in future.