Abstract
Introns near 5′ end of genes generally enhance gene expression because of an enhancer /a promoter within their sequence or as intron-mediated enhancement. Surprisingly, our previous experiments found that the vector containing the last intron (intron V) of human thromobopoietin (hTPO) expressed higher hTPO in cos-1 cell than the vector containing intron I regulated by cytomegalovirus promoter. Moreover, regulated by 1.0 kb rat whey acidic protein promoter, hTPO expression was higher in transgenic mice generated by intron V-TPOcDNA than in transgenic mice generated by TPOcDNA and TPOgDNA. However, it is unknown whether the enhancement of hTPO expression by intron I is decreased by uAUG7 at 5′-UTR of hTPO in vivo. Currently, we constructed vectors regulated by stronger 6.5kb β-casein promoter, including pTPOGA (containing TPOcDNA), pTPOGB (containing TUR-TPOcDNA, TUR including exon1, intron I and non-coding exon2 of hTPO gene), pTPOGC (containing ΔTUR-TPOcDNA, nucleotides of TUR from uAUG7 to physiological AUG were deleted), pTPOGD (containing intron V-TPOcDNA) and pTPOGE (containing TPOgDNA), to evaluate the effect of intron I on hTPO expression and to further verify whether intron V enhances hTPO expression in the milk of transgenic mice. The results demonstrated that intron V, not intron I improved hTPO expression.
Highlights
Introns near 5′ end of genes generally enhance gene expression because of an enhancer /a promoter within their sequence or as intron-mediated enhancement
It is still uncertain whether there exist other specific elements in Human thrombopoietin (hTPO) gene, which may enhance hTPO expression level when the body is stimulated by external factors
In order to eliminate the effect of uAUG7 on intron I influence on the expression of hTPO gene, Δ TUR was deleted from uAUG7 to physiological AUG to at TUR, but still contained the correct splicing sequence of introns and exons (Fig. 1b)
Summary
Introns near 5′ end of genes generally enhance gene expression because of an enhancer /a promoter within their sequence or as intron-mediated enhancement. In our previous study, we found that the last intron (intron V) rather than the first intron significantly improved hTPO expression in cos-1 cell[13] and the hTPO expression was more higher in the milk of transgenic mice carrying intron V-TPOcDNA than in transgenic mice carrying TPOcDNA and TPOgDNA under the control of 1.0 kb rat WAP promoter[14] It is interesting but still unknown whether the effect of the first intron on enhancement of hTPO gene expression is weaken or neutralized by the presence of uAUGs at 5′ -UTR of hTPO gene in vivo. We speculated that the last intron probably enhance hTPO expression in the milk of transgenic animals, especially large transgenic animals, which would be benefit for producing abundant recombinant hTPO with natural structure in therapeutic application
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