Title

Towards high levels of COL1A1 transgene expression

Date of Completion

January 1997

Keywords

Biology, Molecular

Degree

Ph.D.

Abstract

Knowledge of the elements required to achieve endogenous levels of COL1A1 expression is important to our understanding of gene regulation, in general, and for the development of gene therapeutic strategies applicable to the genetic disorder Osteogenesis Imperfecta, in particular. While the human COL1A1 minigene is expressed at levels equivalent to the endogenous gene, little progress has been made towards understanding the elements required for this expression.^ Our laboratory has been studying the regulation of the rat COL1A1 gene utilizing two families of transgenes, designated ColCAT and pOBColCAT. We chose to re-derive ColCAT, which relies on the SV40 t-Ag splicing unit, in a new generation of expression vectors, pOBCAT, to eliminate the problem of cryptic splice site selection. pOBColCAT includes the COL1A1 first exon and most of the first intron and utilizes the COL1A1 splice donor and the SV40 16S splice acceptor as the splicing unit. Stable transfection of pOBColCAT into the osteoblastic cell line Pyla results in a 50-100 fold increase in CAT activity as compared with ColCAT. This increase probably results from replacing the SV40 t-Ag splicing unit with a more efficient unit. The potential for transcriptional enhancement from the COL1A1 first intron was addressed by replacing this sequence with the entire SV40 16S splicing unit. This new construct was expressed at levels equivalent to pOBColCAT.^ Despite high levels of activity, pOBColCAT is expressed at lower levels than the endogenous COL1A1 gene suggesting it does not contain all of the essential regulatory elements. To identify addition elements required for high levels of expression, we chose to introduce two major regions of the human COL1A1 minigene into pOBColCAT: the 3$\sp\prime$ End and the minigene Body. Both regions were assessed for transcriptional enhancing activity and for effects on mRNA stability. Analysis of the minigene Body revealed an orientation-independent five fold increase in CAT activity. In contrast the 3$\sp\prime$ End showed a modest 61% increase in CAT activity. Neither region increased the mRNA half-life of the parent construct, pOBColCAT, suggesting that CAT-specific mRNA instability elements serve as dominant negative regulators of stability. ^

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