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Ferric Chelate Reductase Knockdown in Drosophila S2 Cells
Murphy, Laura
Holst, Jessica
Kane, Gregory
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Denver, CO
Metropolitan State University of Denver
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Conference Papers ( sobekcm )


Collected for Auraria Institutional Repository by the Self-Submittal tool. Submitted by Matthew Mariner.
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Faculty mentors: Emily Ragan, Maureen Gorman
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Major: Chemistry

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Fig . 3 . Standard curve to assess qPCR primer and reaction optimization . Standard curve generated using 10 fold dilution series of S 2 cell cDNA from a negative control dsRNA experiment using dsRNA to GFP . Threshold cycle Cq plotted against the log of the starting quantity of template for each dilution . Regression equations and R 2 values are displayed . The E value for the CG 8399 primers is 97 . 7 % , and the E value for our reference primers is 92 . 8 % . These E values tell us our new CG 8399 nearly doubled after each cycle of amplification . E values of < 100 % reaffirms the absence of primer dimers . A) Fig. 4. CG8399 RNAi gene knockdown data. (A) The delta Ct shows the difference in number of cycles needed to reach a threshold level of DNA. Higher starting amounts of cDNA give lower Ct values. In all cells, the C T for CG8399 cDNA levels (target) are higher than reference gene GAPDH levels, showing the CG8399 is expressed at a lower level than GAPDH even under normal conditions. The difference is even larger in the cells exposed to dsRNA for CG8399. This is consistent with a knockdown in CG8399 mRNA after treatment with CG8399 dsRNA. Average 8399 delta Ct of ( 7.2 0.1924, n=3) (B). Relative gene expression shows a CG8399 fold change of (0.3987 0.30073, n=3, p= 0.001252) as compared to the GFP treated samples. This fold change shows a statistically significant decrease. A similar fold change is seen when 8399 results are compared with untreated cells. Results reported as Mean 95% CI. Laura Murphy, Jessica Holst, Gregory Kane & Emily J. Ragan Department of Chemistry, Metropolitan State University of Denver Introduction . Ferric Chelate Reductase Knockdown in Drosophila S2 Cells Fig . 1 . Proposed mechanism for the uptake of iron into non intestinal Drosophila cells . Potential ferric reductase CG 8399 reduces Fe 3 + to Fe 2 + , which is then transported into the cell via a ferrous iron transporter . Results Methods Acknowledgements Although the mechanisms of cellular iron uptake in mammals are largely understood, there is still little known about the mechanism of iron transport in insects . In insects, iron is necessary for many physiological processes from energy metabolism to detoxification of pesticides . However, ferrous iron can also cause cellular damage through the Fenton Reaction . Due to this, iron needs to be well regulated to maintain balance between necessity and toxicity . Understanding iron metabolism in insects could lead to novel insecticides and a better understanding of iron metabolism in humans . In mammals, a duodenal cytochrome b acts as a ferric reductase to allow for uptake of iron into intestinal cells . We identified a cytochrome b 561 family member in Drosophila , CG 8399 , that is expressed in Drosophila S 2 cells . CG 8399 is similar to ferric chelate reductase 1 in humans as both contain reeler , domon and cytochrome b 561 domains . We are testing the hypothesis that there is reduction of iron by CG 8399 , which allows the transport of iron into the cells . To test this hypothesis, we used RNA interference (RNAi) to knockdown the CG 8399 mRNA . We quantified the RNAi knockdown caused by RNAi using real time PCR . We designed new primers for a different RNAi experiment to see if we can obtain an even larger knockdown . We will compare iron concentrations between untreated and treated cells using a ferrozine based iron content assay . A lower concentration of iron in CG 8399 RNAi treated cells would support our hypothesis . Discussion Conclusion and Future Plans Double stranded RNA (dsRNA) synthesis and RNA interference RNAi synthesis PCR primers with T7 overhang designed using eRNAi (older primers) or SnapDragon PCR products analyzed with 1% agarose gel ethidium bromide gel 10 ug of purified PCR product was used for dsRNA synthesis using MEGAscript RNAi kit ( Ambion ) For RNAi we used 1.0 x 10 7 Added 75µg of dsRNA to each flask. Incubated the flask at 24 C for three days. RNA was isolated from the cells on day four using PARIS kit (Thermo Fisher) Purified RNA was treated with 1 µL recombinant DNase I (Thermo Fisher) to remove genomic DNA, then reverse transcribed to cDNA and purified using the GeneJET RNA purification kit (Thermo Fisher). Thank you to our collaborator, Dr . Maureen Gorman, at Kansas State University . Thank you to Omar Rodriguez to assistance with the qPCR standard curve . Research supported by funding from NSF (IOS 1 656407 , Ragan and 1656388 , Gorman) . Sg 4 cells from Drosophila Genomics Resource Center (DGRC), which is supported by NIH grant 2 P 40 OD 010949 . CG8399 RB (FBgn0034067) (2342 bp) Figure 5: 8399 cDNA sequence and primer sites for dsRNA synthesis and qPCR analysis of RNAi mediated gene silencing . An exon map of CG8399 isoform RB is shown. The only other CG8399 isoform, RA, is missing a small part of the beginning of exon 7, shown in pink. The primers used for past CG8399 dsRNA synthesis are shown as purple triangles below exon 2 and exon 4 with the region they amplify in purple. The primers used for past qPCR are shown in orange. New primers for amplification for RNAi were designed and are shown in gray below exons 5 and 6. The product from PCR amplification using those new primers is shown in Figure 6. Quantitative PCR (qPCR) 2 µL of 1.0 ng/µL cDNA was added to each well (96 well plate) with 18 µL master mix (cDNA forward and reverse primers, nuclease free water and 2x SsoAdvanced SYBR Green Supermix , Bio Rad) using qPCR conditions of 40 cycles, 60 ° C annealing temp, melt curve added. qPCR assay optimization performed on CG8399 cDNA and GAPDH primers using GFP treated cDNA in 10 fold dilution series, then cDNA from RNAi with CG8399 was analyzed Data analyzed using CT method. For our experiment, Average C T = Average of 3 biological replicates, C T = Average C T (GAPDH primers) Average C T (8399 primers); C T = C T (test) C T (control) where the test is 8399 dsRNA treatment and the control is either GFP treated cells or untreated cells. 2 CT = relative gene expression fold change Fig. 2 RNA interference (RNAi) Mechanism Double stranded RNA (dsRNA) is absorbed by S2 cells via endocytosis, cleaved into ~21 nt fragments by a protein called dicer, and incorporated in the RNA induced silencing complex ( RISC). RISC degrades mRNA matching the 21 nt portions of dsRNA. The degradation of mRNA matching the added RNA prevents the mRNA from being translated into protein. In our experiment we are adding dsRNA for CG8399 to cause degradation of CG8399 mRNA and prevent CG8399 protein production. Figure 6 : Agarose Gel of CG 8399 PCR Product . A garose gel of PCR product using new CG 8399 primers . The gel shows a band for the CG 8399 product at the expected 615 bp . The CG 8399 product shows a defined signal without trace of primer dimers . . The fold change for CG 8399 expression after RNAi shown in figure 4 is about 0 . 4 . We have performed a similar RNAi experiment with another putative ferric reductase, CG 1275 , and achieved a lower fold change of 0 . 1 0 . 3 for CG 1275 knockdown in multiple experiments . The fold change correlates to how much expression of CG 8399 or CG 1275 remains after knockdown of mRNA through RNAi . The lower the fold change, the better the knockdown . Thus the knockdown of CG 8399 has been less successful and less consistent than that of CG 1275 . Therefore, we redesigned the dsRNA primers for CG 8399 , as shown in figure 5 . The CG 8399 primers have been redesigned to target a different region than the previous dsRNA primers and spanning a slightly larger portion of CG 8399 . We hope the new RNAi region will give a more definitive knockdown . The differences in knockdown and fold change of CG 8399 between the newly developed and previously used dsRNA primers need to be compared . After determining which regions are more successful in RNA interference, we will select the one which provides the best knockdown for use in future RNAi experiments . It is important that we are able to effectively and consistently knockdown the potential putative ferric reductases so that we can then accurately analyze the iron content of the cells following RNAi . At that point we will be able to test our hypothesis that the levels of iron will be decreased in the CG 8399 RNAi treated cells . qPCR CG8399 CG8399 RB (FBgn0034067) (2342 bp) B) B)