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-specific sequences needed for transcriptional activation. The elements required 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 activity. Using stably transfected osteoblastic Py1a cells, we studied a series of constructs (pOBColCAT) designed to characterize further the elements required for high level of expression. pOBColCAT, which contains the COL1A1 first intron, was expressed at 50-100-fold higher levels than ColCAT 3.6, which lacks the first intron. This difference is best explained by improved mRNA processing rather than a transcriptional effect. Furthermore, variation in activity observed with the intron deletion constructs is best explained by altered mRNA splicing. Two major regions of the human COL1A1 minigene, the 3'-flanking sequences and the minigene body, were introduced into pOBColCAT to assess both transcriptional enhancing activity and the effect on mRNA stability. Analysis of the minigene body, which includes the first five exons and introns fused with the terminal six introns and exons, revealed an orientation-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 dominant negative regulators of stability. This study suggests that other sites within the body of the COL1A1 minigene are important for high expression, e.g. during periods of rapid extracellular matrix production.
Breault
D T
DT
Department of Pediatrics, University of Connecticut Health Center, Farmington, Connecticut 06030-1515, USA.
Lichtler
A C
AC
Rowe
D W
DW
D000818:Animals; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003094:Collagen / Q000235:genetics*; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D007438:Introns; D010006:Osteoblasts / Q000378:metabolism*; D011347:Procollagen / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D014162:Transfection; D019076:Transgenes
D011347:Procollagen; D012333:RNA, Messenger; D003094:Collagen; D015500:Chloramphenicol O-Acetyltransferase