BACKGROUND: Intronic sequences have the potential to improve gene expression in eukaryotes by a variety of mechanisms. In this context, human beta-globin (hBG) introns were inserted into the human factor IX (hFIX) cDNA in cytomegalovirus (CMV)-regulated plasmids. The resulting construct was then used for further expression analysis in vitro. METHODS: Seven hFIX-expressing plasmids with different combinations of the two hBG introns and the Kozak element were constructed and used for a systematic expression analysis in cultured Chinese hamster ovary (CHO) cells. In parallel, the hBG intronic sequences were analysed for the presence of possible regulatory elements. RESULTS: All the constructed plasmids resulted in transient expression of the hFIX. However, the coagulation activities varied according to the particular constructs used. Based on the hFIX antigenic assay, a wide range of variation was observed during persistent expression. The second hBG intron appears to be more effective than the first one. The expression level was further increased upon the inclusion of the Kozak element. Sequence analysis has detected several transcription factor binding (TFB) motifs in both of the introns, but with a higher frequency in the second one. CONCLUSIONS: Potentials of hBG introns as enhancer-like elements for the expression of the hFIX in cultured CHO cells and a higher activity with respect to the second hBG intron compared to the first one were demonstrated. The larger number of TFBs in the second hBG intron reflects its stronger effect. The results obtained suggest possible synergistic functions of the hBG introns and Kozak on the expression level of hFIX in vitro.
BACKGROUND: Intronic sequences have the potential to improve gene expression in eukaryotes by a variety of mechanisms. In this context, humanbeta-globin (hBG) introns were inserted into the human factor IX (hFIX) cDNA in cytomegalovirus (CMV)-regulated plasmids. The resulting construct was then used for further expression analysis in vitro. METHODS: Seven hFIX-expressing plasmids with different combinations of the two hBG introns and the Kozak element were constructed and used for a systematic expression analysis in cultured Chinese hamster ovary (CHO) cells. In parallel, the hBG intronic sequences were analysed for the presence of possible regulatory elements. RESULTS: All the constructed plasmids resulted in transient expression of the hFIX. However, the coagulation activities varied according to the particular constructs used. Based on the hFIX antigenic assay, a wide range of variation was observed during persistent expression. The second hBG intron appears to be more effective than the first one. The expression level was further increased upon the inclusion of the Kozak element. Sequence analysis has detected several transcription factor binding (TFB) motifs in both of the introns, but with a higher frequency in the second one. CONCLUSIONS: Potentials of hBG introns as enhancer-like elements for the expression of the hFIX in cultured CHO cells and a higher activity with respect to the second hBG intron compared to the first one were demonstrated. The larger number of TFBs in the second hBG intron reflects its stronger effect. The results obtained suggest possible synergistic functions of the hBG introns and Kozak on the expression level of hFIX in vitro.
Authors: Vandhana Muralidharan-Chari; Zachary Wurz; Francis Doyle; Matthew Henry; Andreas Diendorfer; Scott A Tenenbaum; Nicole Borth; Edward Eveleth; Susan T Sharfstein Journal: J Biotechnol Date: 2020-10-25 Impact factor: 3.307