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Examination of the regulatory activity of a selected haplotype-block via an enhancer-reporter assay
Lucas Schröfl
Kirsten Senti
Barbara Wallner
Master thesis - University of Veterinary Medicine Vienna - 2020
The lab of Christian Schlötterer conducted experimental evolution studies with a South African population of Drosophila simulans for 60 non-overlapping generations under stressful, hot-cycling (18°C/28°C) environmental conditions. The most significant and focal allele frequency change was observed for SNPs located in a putative cis-regulatory structures of the gene GD13851. This suggests that modulated gene expression of GD13851 may be the causative adaptive change leading to increased thermal fitness in hot environments. To test if the SNPs in the particular cis-regulatory region cause a change in the spatiotemporal transcription regulation of GD13851, enhancer-reporter assays in D. simulans were performed. Therefore multiple enhancer-reporter vectors were generated, in which the reporter gene lacZ is driven from a minimal promoter, by the putative cis-regulatory sequence of either the “evolved” or the ancestral haplotype. Additionally, this enhancer-reporter plasmid contains an attB site to enforce stable, site-specific genomic integration via the ɸ-C31 integrase system. As a result position effects on gene expression are considered constant, and differences in spatio-temporal expression of the reporter gene can be considered as an inherent feature of the different enhancer fragments. The propagation of these constructs was performed using NEB® Stable Competent Escherichia coli cells, a strain that is deficient in the recA gene, a gene that has been proven to be central to recombination processes in E. coli. Surprisingly, prolonged cultivation resulted in this strain in intramolecular plasmid recombination within sub-elements of our enhancer-reporter vectors. The recE pathway could be a mean to facilitate recombination in recA mutant E. coli. Microinjections into an attP site carrying D. simulans (y-,w-) strain were executed. A mini-white gene present in the enhancer-reporter construct renders screening and identification of successful integration events straightforward in the w- genetic background. Unfortunately, no transformed F1 offspring was received for any of the 6 injected constructs. The presence of the attP site in the D. simulans strain was validated by PCR. The vasa promoter from D. melanogaster that was used in order to drive ɸ-C31 integrase is suspected to be non-functional in Drosophila simulans.
Masterarbeit - Veterinärmedizinische Universität Wien - 2020
Englisch
2020
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