An analysis of alpha(1)-proteinase inhibitor gene regulatory elements utilizing transgenic mice

Abstract

The {dollar}\alpha\sb1{dollar}-antitrypsin gene in humans and its murine counterpart the {dollar}\alpha\sb1{dollar}-proteinase inhibitor genes ({dollar}\alpha\sb1{dollar}-PI) are examples of transcriptionally regulated, liver abundant genes. Mutation of the gene in humans has been shown to result in early onset of emphysema. A murine model for this disease would require a thorough understanding of the elements regulating the expression of these genes. Previous work in hepatoma cell lines has identified multiple regulatory elements within 523 bp of the murine {dollar}\alpha\sb1{dollar}-PI transcriptional start site. The studies in this thesis were designed to determine if these elements were sufficient to regulate gene expression in a similar manner in vivo. To this end I established 31 transgenic lines containing various DNA constructs which test the in vivo function of several of the 5{dollar}\sp\prime{dollar} end cis-acting DNA sequence elements. Each construct includes from 300 to 10,000 bp of {dollar}\alpha\sb1{dollar}-PI 5{dollar}\sp\prime{dollar} sequence linked to either a human growth hormone releasing factor minigene reporter or to a mouse {dollar}\alpha\sb1{dollar}-PI minigene construct. Expression of the constructs was determined at 3 levels of sensitivity using reverse transcription PCR, RNAse protection and Northern hybridization. Twenty six lines were generated from the {dollar}\alpha\sb1{dollar}-PI driven GRF constructs. Only 4 lines showed the expected size bands in the RNAse protection assays. On the other hand, 2 out of 3 independent {dollar}\alpha\sb1{dollar} -PI minigene lines expressed the minigene at levels detectable by RNAse protection. None of the lines produced a detectable signal by Northern. I conclude that although the 5{dollar}\sp\prime{dollar} regulatory elements used in this study were capable of directing high levels of expression when transiently transfected into tissue culture cell lines, they were not sufficient to regulate expression of the reporter gene after integration into the murine genome. The expression of the {dollar}\alpha\sb1{dollar}-PI minigene construct at levels higher than any of the GRF constructs indicates that some of the elements necessary for expression of the {dollar}\alpha\sb1{dollar}-PI genes reside in either one of the introns present in the construct or in the 3{dollar}\sp\prime{dollar} sequence.