Dissertation/Thesis Abstract

Differential RNAi Approaches to Enhance Knockdown Efficiency of Target Gene Transcripts in the Highly DDT-Resistant 91-R Strain of Drosophila melanogaster
by Mazgaeen, Lalita, M.S., Southern Illinois University at Edwardsville, 2017, 77; 10274565
Abstract (Summary)

The aim of this study was to demonstrate the enhanced down-regulation of tissue-specific and hard to knockdown Cyp4g1 gene known to be associated with DDT-resistance in highly DDT-resistance 91-R strain of Drosophila melanogaster by utilizing double-stranded RNA (dsRNA) combined with different polymeric nanoparticles that were delivered through feeding. 91-R female flies were fed with dsRNA complexed with polymeric nanoparticles (e.g., chitosan, polyamidoamine (PAMAM) dendrimers, and PAMAM dendrimer-modified selenium nanoparticles). Behavioral responses were, then, assessed through contact exposure bioassay with DDT (1 mg/vial) to demonstrate increased sensitivity of 91-R female flies to DDT. Also, quantitative PCRs were performed to determine altered transcript levels of the target genes. Nanoparticle-Cyp4g1 dsRNA-fed and nanoparicle-Mrp1 dsRNA-fed 91-R females exhibited significantly increased knockdown sensitivity (KS) compared to nanoparticle-Rp49 dsRNA-fed 91-R females. Likewise, 91-R female flies fed with, chitosan (CN)-selenium (SN)-Mrp1 dsRNA, PAMAM (PDN)-SN- Mrp1 ds RNA, CN-Mrp1dsRNA showed significantly increased KS (54.0, 45.39 and 43.21 %, respectively), compared to those flies fed with nanoparticle-Rp49 dsRNA. 91-R adult females treated with PDN-Cyp4g1 dsRNA, PDN-SN-Cyp4g1 dsRNA and CN-SN-Cyp4g1 significantly increased KS (49.88, 40.27 and 27.24 %, respectively), compared to 91-R females treated with PDN-Rp49, PDN-SN-Rp49 and CN-SN- Rp49 dsRNAs, respectively. However, feeding dsRNAs alone ( Mrp1, Cyp4g1, or Rp49 dsRNAs) did not change the knockdown behavior of flies (χ2 = 4.8, df = 6, p < 0.01). <p> Significantly increased KS in nanoparticle-dsRNA fed flies suggests successful delivery of Cyp4g1 and Mrp1 dsRNA into the target cells’ cytoplasm where Dicer can further process dsRNA molecules. Interestingly, transcript level of Cyp4g1 gene was significantly reduced (59 %) only in CN-SN-Cyp4g1-fed female flies. Similarly, 91-R females fed with CN-SN-Mrp1 exhibited significantly reduced (76 %) Mrp1 transcripts compared to CN-SN- Rp49-fed flies. These findings from qPCRs, however, shouldn’t be used as final records to explain increased KS reported in this study since 91-R females fed with other nanoparticle-dsRNA complexes produced inconclusive results. Further research is necessary to address the inconclusive results from qPCR analysis.

Present research demonstrates the usefulness of nanoparticles in RNAi. Particularly, this technique may be useful to validate linkage and function of two or more genes associated with DDT resistance.

Indexing (document details)
Advisor: Yoon, Kyong-Sup
Commitee: Lin, Zhi-Qing, Theodorakis, Chris
School: Southern Illinois University at Edwardsville
Department: Environmental Sciences
School Location: United States -- Illinois
Source: MAI 56/04M(E), Masters Abstracts International
Subjects: Environmental science
Keywords: Ddt, Drosophila melanogaster, Nanoparticles, RNAi, Resistance
Publication Number: 10274565
ISBN: 978-1-369-81404-0
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