COL1A1 TRANSGENE EXPRESSION IN STABLY TRANSFECTED OSTEOBLASTIC CELLS - RELATIVE CONTRIBUTIONS OF FIRST INTRON, 3'-FLANKING SEQUENCES, AND SEQUENCES DERIVED FROM THE BODY OF THE HUMAN COL1A1 MINIGENE

Collagen reporter gene constructs have be used to identify cell-specif ic sequences needed for transcriptional activation, The elements requi red for endogenous levels of COL1A1 expression, however, have not been elucidated, The human COL1A1 minigene is expressed at high levels and likely harbors sequence elements required for endogenous levels of ac tivity, Using stably transfected osteoblastic Pyla cells, me studied a series of constructs (pOBColCAT) designed to characterize further the elements required for high level of expression. pOBColCAT, which cont ains the COL1A1 first intron, was expressed at 50-100-fold higher leve ls than ColCAT 3.6, which lacks the first intron. This difference is b est explained by improved mRNA processing rather than a transcriptiona l effect, Furthermore, variation in activity observed with the intron deletion constructs is best explained by altered mRNA splicing, Two ma jor regions of the human COL1A1 minigene, the 3'-flanking sequences an d the minigene body, were introduced into pOBColCAT to assess both tra nscriptional enhancing activity and the effect on mRNA stability, Anal ysis of the minigene body, which includes the first five exons and int rons fused with the terminal six introns and exons, revealed an orient ation-independent 5-fold increase in CAT activity, In contrast the 3'- flanking sequences gave rise to a modest 61% increase in CAT activity, Neither region increased the mRNA half-life of the parent construct, suggesting that CAT-specific mRNA instability elements may serve as do minant negative regulators of stability. This study suggests that othe r sites within the body of the COL1A1 minigene are important for high expression, e.g. during periods of rapid extracellular matrix producti on.