Design and Preliminary Assembly of Custom TALEN Constructs for DNA Targeting of Sphinx 1/2, a Drosophila Serine Protease Homologue
Harrison, Sally A.
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In response to microbial infection, Drosophila melanogaster initiates an immune response leading to defense mechanisms, triggering the Toll pathway via Gram-positive bacterial infection or fungal infection. This results in the cleavage of the protein, Spatzle to activate the transcription of the Toll gene. The proteases and ligands in between these beginning and ending steps have been an important area of study to fully understand the complexity of Drosophila’s innate immunity. There are still a few unknown serine proteases that need further analysis and testing – one being Sphinx 1/2, a serine protease homolog. After the mapping of the entire Drosophila genome, new tools have been discovered for genome engineering. The present study focused on the early construction of plasmid vectors targeting the Sphinx 1/2 gene through use of a relatively new technique for modifying DNA using transcription activator-like effector nucleases (TALENs). This technique takes advantage of the TALEN’s ability to target any gene in the Drosophila genome through use of repeat variable di-residues. The results show the completion of the first step of TALEN unit assembly. Verification of designed primers led to the proper TALEN design, targeting Sphinx 1/2. Utilizing the Golden Gate cloning strategy, six specific repeat variable di-residues for three different vectors were successfully assembled for each side of the TALEN for both Sphinx 1 and Sphinx 2. The vectors were verified by colony PCR followed by digestion to visualize the different PCR fragments to verify where a restriction enzyme would cut in the sequence. The results indicated that further testing could be done on Sphinx 1/2 through mutant flies to test its greater role in the Toll pathway. This study also proved the effectiveness of TALENs in manipulating the Drosophila genome and perhaps for genes outside of Drosophila.