38th Annual Meeting of the Society for Invertebrate Pathology

Transgenesis and paratransgenesis offer powerful approaches for analysis of molecular interactions between arthropod vectors, their microbial symbionts, and human pathogens. We addressed the need for transformation systems for the study of ticks and their associated microbes by exploring the ability of the Sleeping Beauty transformation system, a reconstructed Tc1/mariner related transposable element and a reconstructed transposase from teleost fish sequences (Ivics et al. 1997 Cell 91:501), to mediate transformation of ixodid tick cells. Cell line ISE6 from the black legged tick, Ixodes scapularis, was stably transformed using marked Sleeping Beauty transposons in the presence of plasmids expressing transposase. Marker genes were either red fluorescent protein (DsRed2) or neomycin resistance genes. Transient expression of DsRed2 lasted 4 weeks and by 6 weeks approximately 90% of the cells lost expression of DsRed2. The remaining positive cells were stably transfected, and selectable using a neomycin analog, G418. Cloning and sequencing of the integration sites demonstrated that insertions of the DsRed2 gene within the cells’ genome occurred via the action of the Sleeping Beauty transposase. This system has potential for functional genetic analysis of interactions between ticks and microorganisms.