Transgenic Rescue of Ce-daf-16 with Bma-daf-16 Alanna Gould, Matthew Schulgit (Kirsten Crossgrove) University of Wisconsin – Whitewater, Department of Biological Sciences Abstract Brugia malayi is a parasitic nematode which causes lymphatic filariasis in humans when adults accumulate in the lymphatic system. Infective stage larvae (iL3) enter the human host via a mosquito bite and molt into L4 larvae. The free-living nematode Caenorhabditis elegans is a model organism which can be useful in the study of B. malayi. The arrested dauer stage in C. elegans is similar to the iL3 stage in B. malayi, and both require environmental signals to exit these stages. DAF-16 is a forkhead transcription factor that binds to DNA to regulate gene expression and is negatively regulated by the insulin/IGF-1 signaling (IIS) pathway. Targets of DAF-16 include many of the genes important for dauer formation in C. elegans. To test whether Bma-DAF-16 functions similarly to Ce-DAF-16, we have cloned the Ce-daf-16 promoter and will clone Bma-daf-16 into a plasmid which will be injected into C. elegans daf-2;daf-16 double mutants, which cannot form dauers. Transgenic worms will be scored based on whether the ability to form dauers has been restored by the presence of Bma-daf-16. We predict that, if Bma-DAF-16 functions the same way as Ce-DAF-16, most or all of the transgenic worms will form dauers. If this does occur, we will demonstrate a shared role for DAF-16 in B. malayi and C. elegans. This will provide a foundation to apply what is known about the role of DAF-16 in regulation of dauer in C. elegans to the role of DAF-16 in regulation of the iL3 stage in B. malayi. This knowledge could then be applied to research on treatment of B. malayi infection. Research Question: Can Bma-daf-16 rescue Ce-daf-16 function in dauer formation? Generation of Transgenic Worms C. elegans daf-2;daf-16 double mutants will be injected with the construct and the pRF4 plasmid which encodes the rol-6 gene. Injected worms will lay eggs and progeny that express the roller phenotype will be used to establish transgenic lines. Approach: Construct transgenesis vector that expresses Bma-daf-16, use construct to make transgenic worms, and assess Bma-daf-16 ability to rescue Ce-daf-16 function using dauer switching assays. Once transgenic lines have been established, we will use fluorescent microscopy to asses GFP expression patterns. This will tell us whether Bma-daf-16 is being expressed in the right places and at the right time. Cloning of Bma-daf-16 to Transgenesis Vector Ladder cDNA promoter Figure 4. Electrophoresis gel showing amplified Bma-daf-16 cDNA and Ce-daf-16 promoter products. We used the polymerase chain reaction (PCR) to amplify the Bma-daf-16 cDNA and Ce-daf-16 promoter. Figure 7. Microinjection of C. elegans. Image source: http://post.queensu.ca/~chinsang/links.html Dauer Switching Assays We will perform three trials to determine the proportion of dauer vs non-dauer individuals, which will then be compared to the proportion from the daf-2;daf-16 parental stock using a chi square test. We engineered restriction enzyme sites to facilitate cloning of the cDNA and promoter into the transgenesis plasmid. The final construct containing the promoter, cDNA, and green fluorescent protein (GFP) will be used to create transgenic worms. The Ce-daf-16 promoter will cause Bma-daf-16 to be expressed at the right time and place and GFP will allow us to see where it is expressed. Transgenesis vector We predict that, if Bma-DAF-16 restores the ability to form dauers, no parental daf-2;daf-16 mutants will form dauers while most or all of the transgenic worms will form dauers. Figure 8. C. elegans dauer larva and reproductive adult. Adapted from: iamphioxus.org Introduction Conclusions and Future Directions The Ce-daf-16 promoter and Bma-daf-16 cDNA were amplified The promoter and cDNA were sub-cloned to the pGEM T easy vector The Ce-daf-16 promoter sequence was confirmed Currently, we are working on ligating the promoter to the transgenesis vector and will need to sequence, digest, and ligate the cDNA to the vector as well. Once cloning is finished we will perform microinjections of the construct on C. elegans daf-2;daf-16 double mutants and select progeny with which to establish transgenic lines. The transgenic lines will be used in dauer switching assays. Figure 1. Life cycle of B. malayi. Image source: cdc.org Figure 2. Life cycle of C. elegans. Image source: Wormatlas.org B. malayi and C. elegans undergo similar molts and life cycle stages. It is thought that exit from the iL3 stage in B. malayi is similar to exit from dauer in C. elegans (Crook 2014). The initial amplification was successful, but following the ligation of the Ce-daf-16 promoter PCR product and pPD95.75 plasmid, no clones were isolated. This could be due to difficulty in restriction enzymes cutting at the end of a PCR product. Thus, we cloned the Ce-daf-16 promoter and Bma-daf-16 cDNA into a T vector (easy cloning of PCR products) to improve the efficiency of the restriction enzyme digestion. Acknowledgements We thank the UWW Undergraduate Research Program and Department of Biological Sciences for funding the project, and members of the Crossgrove lab for general assistance and support. Works Cited Crook, M. (2014) The dauer hypothesis and the evolution of parasitism: 20 years on and still going strong. International Journal of Parasitology 44: 1-8. Hung, W.L., Wang, Y., Chitturi, J., Zhen, M. (2014). A Caenorhabditis elegans developmental decision requires insulin signaling-mediated neuron-intestine communication. Development 141: 1767-1779. Lee, S.S., Kennedy, S., Tolonen, A. C., Ruvkun, G. (2003) DAF-16 target genes that control C. elegans life-span and metabolism. Science 300: 644-647. Murphy, C.T., Hu, P.J. (2013) Insulin/Insulin-like growth factor signaling in C. elegans. Wormbook, ed. The C. elegans Research Committee. doi/10.1895/wormbook.1.164.1 DAF-16 regulates the expression of genes necessary for dauer formation (Lee et al, 2003). In the absence of insulin signaling DAF-16 is active and worms form dauers (Murphy and Hu, 2013). M 1 2 3 4 5 6 Ladder promoter * * Figure 5. Electrophoresis gel of digested Ce-daf-16 promoter. This clone was sequenced and confirmed to be the correct clone. Figure 3. IIS pathway. Adapted from: Wesley, et al. (2014) Hypothesis Bma-DAF-16 functions in regulation of iL3 similarly to Ce-DAF-16 in regulation of dauer. Figure 6. Electrophoresis gel of digested Bma-daf-16 cDNA. The lanes 3 and 5 were identified as possible positive clones and will be sequenced to confirm their identity.