Our Research Methods Keyata Lewis, Jessica Holst, Gregory Kane, Mirella Castaneda, Maureen Gorman, Emily Ragan Department of Chemistry and Biochemistry, Metropolitan State University of Denver Putative Ferric Reductases CG1275 and nemy in Iron Uptake in Drosophila S2 cells Primer selection Followed up on work by former student Mirella Castaneda. Picked regions common to all nemy isoforms (see figure 2). Used PrimerQuest Tool to design primers for RNAi and qPCR regions, and ordered primers from IDT Standard PCR Used 1.16ng/ÂµL cDNA extracted from S2 cells as template DNA. . Created 10 uL reverse and forward Nemy and GAPDH primers dilutions (1:10). Used 25 uL of Master Mix (Promega) by combining forward and reverse primers with PCR reagents. To prepare final samples 1.16ng/ÂµL cDNA was combined with nuclease free H 2 O and Master Mix. Samples were placed in PCR Thermocycler and ran under the protocol: 1.) 2 minutes at 94 Â° C, 2.) 30 seconds at 94 Â° C, 3.) 30 seconds at 52 Â° C, 4.) 30 seconds at 72 Â° C, 5.) 5 minutes at 72 Â° C, 6.) remaining time at 12 Â° C, *Steps 2 4 repeated 29x. Agarose gel electrophoresis Used a 1% agarose gel and 0.5x TBE with ethidium bromide to visualize DNA bands. qPCR primer optimization Prepared serial dilutions from cDNA: 50ng, 5ng, 0.5 ng, 50 pg , 5 pg , and 0.5 pg. Prepared 1:10 dilutions of primers. Created Master Mix by combining appropriate amounts of water, 1:10 primer dilutions, and Syber green mix. Combined appropriate amounts of master mix with appropriate cDNA dilutions. Pipetted 20 uL of sample into wells of a 96 well PCR plate and ran under the following protocol: 1.) 95 Â° C for 3 minutes, 2.) 95 Â° C for10 seconds, 3.) 60 Â° C for 30 seconds, 4.) Repeat 2 Plate Read 39x, 5.) Melt Curve 65 Â° C to 95 Â° C by increments of 0.5 Â° C every 5 seconds. Iron plays key roles in humans and insects, including in the electron transport chain and other electron transport processes. There is still uncertainty about how iron enters the cell in insects 1 . Currently our group is investigating two cytochrome b561 family members, CG1275 and Nemy, as possible ferric reductase components in a Drosophila S2 cell iron uptake pathway. Nemy is known to have ferric reductase properties in Drosophila cells, but its main neurons 2 . Nemy has not yet been directly linked to how flies intake iron, but we do hypothesize that a ferric reductase such as Nemy is needed in order for the cell to uptake iron 2 . We hypothesize that Fe 3+ is reduced to Fe 2+ using a ferric reductase (CG1275 or nemy ), which will then pass through the malvolio protein for entry into the cytosol 2 . Our experiment involves RNA interference (RNAi) gene silencing of CG1275 and nemy . By adding dsRNA with a sequence matching CG1275 or Nemy mRNA, we cause selective degradation of mRNA. After RNAi we extract total RNA and use quantitative PCR to analyze mRNA levels to see if degradation of targeted mRNA has been achieved. We will also measure cellular iron content using a ferrozine based assay. If we detect a decrease in iron content in the cells then we will have support for our hypothesis. S 2 cells (Sg 4 isolate) are from the Drosophila Genomics Resource Center (NIH grant 2 P 40 OD 010949 ) . This research supported by funding from NSF (IOS 1656407 ) . I would also like to thank Dr . Emily Ragan and Dr . Maureen Gorman for organizing, conducting and overseeing all experimentation and research . Lastly like to give thanks to all of my co authors for th eir guidance and assistance . Future plans Analysis of qPCR primers Acknowledgements In the future our group plans to synthesize nemy dsRNA and conduct a RNAi experiment where we add dsRNA that matches nemy to cells, which will cause the degradation of nemy mRNA and, indirectly, nemy protein. After the RNAi experiment, we can then test for successful knockdown of the nemy gene through qPCR and measure iron and protein present in the cells. Our hypothesis is that iron levels in cells would decrease after RNAi mediated gene silencing of nemy . Future experimentation plans are outlined Figure 9. Conclusions I showed that nemy is expressed in the Drosophila S2 cell line at the expected levels. I also gained experience with quantitative PCR by analyzing primers for a target gene, CG1275, which is similar to nemy as well as reference gene primers ( sdha2) . This experience will help me perform the quantitative PCR that will be required at the end of an RNAi experiment to determine if the desired knock down has occurred. The expected ultimate outcome of this experiment is that after the successful interference of RNA in nemy , the cells will have reduced iron uptake. This will support our hypothesis that nemy is a ferric reductase that plays a key role in how the Drosophila melanogaster insect uptakes iron. Through the results of this experiment we can began to conclude how insect cells intake the iron that is used for vital processes. This research could possibly lead to a deeper understanding of the multiple ways that humans can intake iron Figure 1. Hypothetical iron pathway in Drosophila melanogaster cells with Nemy and CG1275 acting as iron reductase. References (1) Bowen, T.; Rubinson , A. C.; Poulson , D. .; Hilse , R. . THE COPPER METABOLISM OF DROSOPHILA. Proc Natl Acad Sci U S A. 1952 , 38 (10), 912 921. (2) Calap Quintana, P.; GonzÃ¡lez FernÃ¡ndez, J.; SebastiÃ¡ Ortega, N.; Llorens , J. V.; MoltÃ³ , M. D. Drosophila Melanogaster Models of Metal Related Human Diseases and Metal Toxicity. Int. J. Mol. Sci. 2017 , 18 (7). https:// doi.org /10.3390/ijms18071456. (3) Lane, D. J. R.; Bae , D. H.; Merlot, A. M.; Sahni , S.; Richardson, D. R. Duodenal Cytochrome b (DCYTB) in Iron Metabolism: An Update on Function and Regulation. Nutrients 2015 , 7 (4), 2274 2296. https:// doi.org /10.3390/nu7042274. (4) Mandilaras , K.; Pathmanathan , T.; Missirlis , F. Iron Absorption in Drosophila Melanogaster. Nutrients 2013 , 5 (5), 1622 1647. https:// doi.org /10.3390/nu5051622. (5) FlyBase . Gene: Dmel \ nemy . FlyBase Gene Report: Dmel \ nemy . Primers and Nemy Expression Figure 5. Gel electrophoresis image of Nemy bands after 30 PCR cycles compared against GAPDH control. Figure 6. Gel electrophoresis image of Nemy bands after 38 PCR cycles compared against GAPDH control. Expression Nemy expression can be detected although lower than GAPDH. During multiple gel electrophoresis trials it was observed that nemy bands were more prominent with increased PCR cycles. For future nemy experimentation lower and more comparative expression than the former control GAPDH Primers Nemy RC isoform was chosen as the sequence to use for primer design because it is one of the shorter sequences. The PrimerQuest Tool was used to design primers for Nemy RC and then primers were ordered from Integrated DNA Technologies. Figure 4. Table containing data for gene expression of GAPDH, CG1275, SDHA and Nemy. Data referenced from FlyBase 5 . Figure 9. Broad overview of future experiments. Future experimentation includes: dsRNA synthesis, RNAi, cell lysis, RNA extraction and qPCR, and measuring cellular protein and iron levels. Figure 7. Amplification Curve for CG1275 and SDHA2 samples. The red line shows the amount of fluorescence used to determine the threshold cycle ( Cq ). Figure 8. Standard Curve for CG1275 and SDHA2 samples. The linear data for SDHA2 suggest it will work well as a reference across a range of starting DNA amounts. Efficiencies (E) are both in the optimal range of 95 105%. Figure 2. FlyBase image of Nemy mRNA transcripts. The (UTR) is in grey on the right, the protein coding region is in 5 Figure 3. Constructed view of Nemy RC gene with primer inserts running from left to right. This image was constructed by importing data from FlyBase in Benchling. 5 The primers for synthesis of dsRNA for an RNAi experiment are in the CDS in blue and light green while the primers for detecting and red.
Hypothetical iron pathway in Drosophila melanogaster cells Drosophila S2 cells iron pathway Human iron pathway
Primer Selection Nemy sequence that can be identified in most of the other isoforms
Primer Synthesis PrimerQuest Tool used to design primers for RNAi Region and qPCR Primers ordered from IDT
Standard PCR & Agarose Gelelectrophoresis
Conclusion & Future Experimentation Conclusion Was able to verify nemy expression in Drosophila S2 cells was at the expected levels Gained experience with quantitative PCR and primer analysis Future Experimentation dsRNA synthesis RNAi Cell lysis and RNAi extraction qPCR Protein and iron content assays