The moderate and sustained increase or decrease in the expression of a single gene can cause disease. Measurement of expression levels for such genes is complicated by several potential observational and biological influences. Recent advances in live animal monitoring provides an opportunity to limit and control for such influences by allowing repeated determination of expression levels in a single animal without the need to sacrifice that animal. These techniques require a signal producing reporter which can be provided in lines of animals by genetic modification. The goal of this work is to develop accurate means of reporting the level of expression of a gene in different tissues of a live animal. We have designed a strategy applicable to any gene which will first be tested on the mouse PGC1? locus given the many known means of regulating its gene expression. We have chosen gene targeting in mouse embryonic stem cells to insert reporters to the target gene to eliminate expression effects common to randomly inserted transgenics. Two reporter genes will be introduced to the mouse PGC1? locus in three different configurations. Accuracy and responsiveness of each reporter/configuration will be empirically determined in ES cells by comparison of reporter output to PGC1? expression levels determined by standard means. Sufficient signal production will be determined in mice and one line with the most faithful and robust reporter output will be used for non-invasive live animal imaging to compare reporter output levels to PGC1? mRNA and protein levels from different tissues. Responsiveness to two treatments known to increase PGC1? in mouse brain will be tested as will the effects of the presence of our Huntington's disease knock-in allele. This exploratory work will provide a test of accuracy of precise reporter models for PGC1? expression and will provide information on needed improvements and pitfalls of whole animal reporter strategies for other genes. The successful production of such accurate reporter mouse lines will accelerate the discovery of means of chronic control of disease gene expression.
We will develop, compare and validate several precise gene expression reporter systems with the goal of making mouse lines that produce quantifiable signal that accurately reflects the expression of a single endogenous gene in live animals. The methods developed in this exploratory work will allow monitoring expression levels of any disease gene and will accelerate the discovery of means of chronic control of disease gene expression.
