[Site directed mutagenesis in ifnbeta gene in order to increasing its mRNA stability and translation level using megaprimer PCR method]

Background and Objectives: Beta interferon [IFNbeta] protein is produced as a recombinant drug and used in treatment of some diseases like Multiple Sclerosis. In eukaryotic cells, IFNbeta mRNA is rapidly degraded and its halflife 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 IFNbeta gene in order to increase its mRNA stability and translational level

Materials and Methods: 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

Results: 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

Conclusion: An 18 nucleotide region of IFNbeta mRNA was deleted. The influence of this microdeletion on mRNA stability and translational efficiency needs to be surveyed in future

New insights into VEGF-A alternative splicing: key regulatory switching in the pathological process

Vascular endothelial growth factor [VEGF-A] is one of the most important regulatory factors in pathological and physiological angiogenesis. Alternative splicing is a complicated molecular process in VEGF-A gene expression which adds complexity to VEGF-A biology. Among all VEGF-A exons, alternative splicing of exon 8 is the key determinant of isoform switching from pro-angiogenic VEGF-xxx to anti-angiogenic VEGF-xxxb. This is known as a key molecular switching in many pathological situations. In fact, the balance between VEGF-xxx and VEGF-xxxb isoforms is a critical controlling switch in both conditions of health and disease. Here, the properties of VEGF-xxx and VEGF-xxxb isoforms were discussed and their regulatory mechanism and their roles in certain pathological processes were evaluated. In summary, it was suggested that C-terminal VEGF-A alternative splicing can provide a new treatment opportunity in angiogenic diseases