Transgenic Rescue of Ce-daf-16 with Bma-daf-16 Melanie Domann (Kirsten Crossgrove) University of Wisconsin – Whitewater, Department of Biological Sciences Abstract Brugia malayi is a parasitic nematode that causes lymphatic filariasis in humans. Infective third larval (iL3) stage parasites are transmitted by mosquitos. Adults accumulate in the lymphatic system and cause disease pathology. The dauer life cycle stage in the free living nematode Caenorhabditis elegans closely resembles the B. malayi iL3 stage. The C. elegans insulin/IGF-1 (IIS) signaling pathway inhibits dauer by negatively regulating the forkhead transcription factor DAF-16. We hypothesize that Bma-DAF-16 functions similarly to Ce-DAF-16 and regulates the formation and maintenance of the iL3 stage. We are testing this by seeing if Bma-daf-16 can rescue Ce-daf-16 mutants. We have successfully amplified the Bma-daf-16b cDNA and Ce-daf-16 promoter using the polymerase chain reaction. We will use the Gibson assembly method to clone both products into a transgenesis vector. If the iL3 stage requires Bma-DAF-16, this may suggest ideas for prevention of B. malayi infection. Research Question: Can Bma-daf-16 rescue Ce-daf-16 function in dauer formation? Generation of Transgenic Worms We will microinject the plasmid into C. elegans daf-2;daf-16 double mutants. Double mutants are necessary because there is no functional insulin receptor in daf-2 mutants. Therefore, they cannot deactivate the transgenic DAF-16 we will be introducing. We will also inject a plasmid which has the rol-6 gene. This gene produces a phenotype that causes the worms to roll around and can become easily identifiable when looking for the transgenic worms. Approach: Inject a transgenic plasmid made up of the Ce-daf-16 promoter and Bma-daf-16 cDNA into C. elegans daf-2;daf-16 double mutants and assess Bma-DAF-16 ability to rescue Ce-DAF-16 function using dauer switching assays. We will identify the transgenic worms by the rolling phenotype and confirm it by using fluorescence microscopy because the Bma-daf-16 will be fused to green fluorescent protein (GFP). Once the worms with the roller phenotype have been identified and confirmed to express GFP, they will be used to establish transgenic lines. Cloning of Bma-daf-16 to Transgenesis Vector Figure 8. Microinjection of C. elegans. Image source: http://post.queensu.ca/~chinsang/links.html Dauer Switching Assays We will have control and experimental transgenic lines with the non-transgenic daf-2;daf-16 worms acting as the controls. We will be raising transgenic (roller) and non-transgenic larvae at 25°C and determining whether they form dauers or not by observing what they look like under the microscope. Figure 4. Gibson Assembly Workflow. Image source: neb.com We are cloning the Bma-daf-16b cDNA and the Ce-daf-16 promoter to the pPD95.75 transgenesis vector using the Gibson assembly method. This method is performed to allow for repair of overlapping DNA molecules in a single step (Gibson et al, 2009). It works by taking a linear vector and overlapping DNA inserts and combining them in a single-tube reaction that contains the Gibson Assembly master mix listed above. This reaction gives a simplified method to forming large DNA molecules from its constituent parts. Figure 9. C. elegans dauer larva and reproductive adult. Adapted from: iamphioxus.org Introduction The non-transgenic worms should not form dauers, but we predict that the transgenic worms will. We will also include a positive control that expresses Ce-daf-16 from a transgene. Three trials will be performed to get an accurate dauer vs. non-dauer count and a chi square test will be used for statistical analysis. Figure 5: Final construct containing the promoter, cDNA, and the transgenesis vector. Image source: neb.com Conclusions and Future Directions We have digested the pPD95.75 transgenesis vector with restriction enzymes and amplified the Ce-daf-16 promoter and Bma-daf-16b cDNA Our next step includes cloning the promoter and the cDNA into the transgenesis plasmid Once we finalize the cloning, we will proceed to the microinjection and dauer switching assays If Bma-DAF-16 can successfully rescue Ce-DAF-16 function, this will suggest a role for insulin signaling in B. malayi and can suggest ideas for prevention of B. malayi infection The Bma-daf-16b cDNA and Ce-daf-16 promoter were amplified using PCR techniques optimized for Gibson assembly. These techniques allowed us to isolate the promoter and cDNA even though the promoter had a size of 6kb. In our previous research, we had difficulty amplifying this large segment of DNA but have now been successful due to this technique. Figure 1. Life cycle of B. malayi. Image source: cdc.org Figure 2. Life cycle of C. elegans. Image source: Wormatlas.org The dauer life cycle stage in C. elegans closely resembles the infective iL3 stage (boxed) in B. malayi (Crook, 2014). Dauer (boxed) is a variant arrest that occurs at the second molt. Dauer arrest can occur from harsh environmental changes (temperature change, amount of food, and population density). The dauer arrest stage in C. elegans is similar to the iL3 stage in B. malayi because both influence the metabolism of the organism and involve the need of specific environmental changes to exit (Hu, P.J. 2007; Crook, 2014). Figure 6: Separate fragments that will be joined into one plasmid. Each color is correlated to Figure 7. 1kb ladder Fig. 7 shows our recent data that includes a 1kb ladder, the transgenesis vector digest with restriction enzymes AgeI and BamHI, the promoter, and the cDNA. The expected sizes for each of these samples are confirmed with this gel data. The next step will be to purify the products and combine the promoter, cDNA, and the vector using the Gibson assembly method. The plasmid will then be sequenced to ensure no errors Acknowledgements We thank the Tri-Beta Research Foundation and the 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. Gibson, D., Young, L., Chuang, R.Y., Venter, J., Hutchison III, C., Smith, H. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 6(5): Hu, P.J., Dauer (August 08, 2007), Wormbook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.144.1, http://www.wormbook.org 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 Ogg, S., Paradis, S., Gottlieb, S., Patterson, G., Lee, L., Tissenbaum, H., Ruvkun, G. (1997) The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 389: 994-999. Wesley, H., Ying, W., Jyothsna, C., Mei, Z. (2014) A Caenorhabditis elegans developmental decision requires insulin signaling-mediated neuron-intestine communication. Development 141: 1767-1779. The insulin/IGF-1 signaling (IIS) pathway is known to regulate dauer with daf-2 encoding the insulin receptor (Murphy et al, 2013). IIS negatively regulates DAF-16, which is a forkhead transcription factor (Ogg et al, 1997). When IIS is turned off, DAF-16 is active, which allows for dauer transformation. Figure 3. IIS pathway. Adapted from: Wesley, et al. (2014) Figure 7: Gel including the transgenesis vector, promoter and cDNA Hypothesis Bma-daf-16 functions similarly to Ce-daf-16 and regulates the formation and maintenance of the iL3 stage.