Design and Preliminary Assembly of Custom TALEN Constructs for DNA Targeting of Sphinx 1/2, a Drosophila serine protease homologue

Abstract

Upon microbial infection, Drosophila melanogaster initiates different signaling pathways to induce immune effector molecules. One such effector molecule, the antimicrobial peptide (AMP), is synthesized upon microbial challenge via gram positive, negative bacterial infection, or fungal infection (Figure 1; right). The Toll Pathway is initiated via challenge by Gram positive bacteria or fungi. The proteolytic cascades involved in this pathway have a functional core of serine proteases (SP) and serine protease homologues (SPH) that are a key component to coagulation of the hemolymph, melanization, and induction of AMPs upon infection. The cleavage of the protein, Spatzle (Spz), the final activator to the transcription of the Toll gene, results from the activation of a SP cascade (Figure 2). While many SPs have been identified and understood in the Toll Pathway, one SPH, Sphinx 1/2, is still poorly understood. Tools for genome engineering, such as transcription activator-like effector nucleases. (TALENs), however, are useful to solve these discrepancies. TALENs are a family of DNA binding proteins that are delivered to host cells, enter the nucleus, and bind to the effector-specific sequences in the host gene promoters to activate transcription. This specialized technique takes advantage of the TALEN’s ability to target any gene in the Drosophila genome through use of repeat variable di-residues (RVDs), tandem repeats of a 34-amino-acid-monomer . In this study, RVDs for the vectors making up each side of the TALENs for Sphinx 1/2 were designed for future testing with a line of mutant D. melanogaster flies to investigate the greater role of this gene in the Toll Pathway.