Browsing by Subject "flex-cassette"

Upregulation of specific helpful proteins represents a possible method for preventing or treating human diseases. Endogenous upregulation (knockup) is the increase of a gene's expression only in cells in which it is already expressed, thus avoiding physiologically abnormal spatiotemporal patterning. A gene's three prime untranslated region (3′UTR) affects protein expression through stability regulation of RNA already transcribed, which suggests 3′UTR modification as a viable route for endogenous upregulation. Mammalian model organisms can be generated in order to test the effects of different 3′UTR modifications, but at great cost of time, effort, and money. If able to predict in advance with an in vitro assay whether an in vivo modification would cause a desirable or undesirable change, these costs could be substantially reduced. In this thesis project, an in vitro assay was used to compare the protein expression influence of twenty neurodegeneration-relevant mouse genes' 3′UTRs to that of a flip-excision cassette (flex-cassette) previously used for in vivo conditional knockup. The assay used was the Promega Dual-Luciferase Reporter Assay, in which plasmids expressing Renilla and Firefly luciferase as reporter and internal control are co-transfected into in vitro cells, then each luciferase's expression measured with its respective substrate and a luminometer. Transfections were carried out in three-well replicates and on multiple days. The aims of the project were the evaluation of the assay's ability to predict in vivo results, the suggestion of 3′UTRs which could be upregulated in vivo by the conditional knockup flex-cassette, and the identification of any trends in 3′UTR-based protein expression influence according to gene function. A number of gene 3′UTRs were identified which were either candidates for flex-cassette upregulation or candidates for use in the flex-cassette to upregulate other genes. However, the flex-cassette's in vitro results were only partially consistent with its previous in vivo results. Specifically, the lox sites in the flex-cassette was observed to lower expression level to a degree not observed in vivo. Additionally, in the course of the project a number of possible workflow improvements were identified, for which suggestions have been made in the text. As such, this in vitro approach requires further study in order to determine suitability for prediction of in vivo 3′UTR behaviour.