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Abstract
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Background Intronic sequences have the potential to improve gene
expression in eukaryotes by a variety of mechanisms. In this context, human
?-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. Copyright ? 2009 John
Wiley
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