ENGINEERING CHO CELLS TO WITHSTAND AMINO ACID STARVATION

Chinese Hamster Ovary (CHO) cells are often times used in the pharmaceutical and bioprocess industry to produce glycosylated protein therapeutics, and are typically grown in serum free media to meet quality control standards. This presents the problem of proteolytic attacks on the final protein product, as the absence of serum also leads to an absence of serum protease inhibitors. It is possible to inhibit protease activity in CHO cells using EDTA and ophenantroline, but it is proposed that RNAi can also be used to inhibit protease activity in CHO cells without permanent alteration of cell genotype. RNAi was used via introducing small interfering RNAs (siRNAs) produced against the protease Matrix Metalloproteinase 14 (MMP14) to test the efficacy of silencing the protease through the use of siRNAs. The most effective siRNAs were then tested via co-transfection with the vector pEGFP-N1 as well as several controls to determine the effect of silencing the MMP14 on the expression of EGFP. It was then determined that some siRNAs were effective in silencing the activity of MMP14 which led to an increase in the activity of EGFP fluorescence. It can be concluded that MMP14 is active in CHO cells and under normal conditions, will attack the EGFP protein, but once silenced, it cannot attack EGFP or other proteins and can therefore lead to an increase in their expression. However, to verify these claims, further testing will need to be performed under conditions of amino acid starvation. A recombinant plasmid utilizing the sequence of the most effective siRNA will need to be created and tested under amino acid starvation conditions to verify the efficacy of RNAi as a silencing mechanism for CHO cells growing in bioreactors