Unknown,Transcriptomics,Genomics,Proteomics

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Expression data from transgenic Drosophila melanogaster adults which contain a knockdown effector of cyp6g1, compared to control flies

ABSTRACT: To test whether other genes were being silenced in the Cyp6g1 knockdown strain due to off-target RNAi effects, and whether other gene expression changes were contributing to the altered susceptibility to imidacloprid in these knockdown flies. A comparison between w;Act5C-GAL4/CyO; UAS:RNAi_Cyp6g1Hp2/TM3Sb and the genetic control w;Act-GAL4/CyO;+/TM3Sb was performed. Ten 2-3 day old adult males or females were transferred to sugar-agar plates and then collected at various time points (0, 2, 5, 8 hours). The RNA samples for up to three independent experiments per timepoint for each genotype were then pooled, in equal concentrations, before hybridisation to the Affymetrix Drosophila GeneChip 1. As a preliminary analysis provided evidence for assuming that there is no interaction between genotype and duration in the sugar-agar plate, the knockdown and control chips for each of the five timepoints provided some degree of replication for the comparison between the knockdown flies and control flies. Differential expression that was consistently observed between knockdown and control over each of the five timepoints was regarded as providing evidence of a genuine difference. 2-3 day old adult flies of the appropriate genotype (Cyp6g1 knockdown or its genetic control) were transferred to bioassay plates and collected after specified timepoints in the assay plate, i.e. 0, 2, 5 or 8 hours. Each experiment contained ?3 replicates of each genotype/timepoint, and a different number of independent experiments (1-3) were pooled per microarray sample as described below. An experiment is defined as treatments set up from the same cohort of flies on a single day. Independent experiments are set up using distinct cohorts of flies, on distinct days. We included one pair of tests to compare samples from single experiments with pooled samples from independent experiments (DS003001 to DS003004, DS003002 to DS003005), and also a single pair of samples to compare males to females (DS003002 to DS003003). As it turned out - in our analysis - the timepoints selected were not discriminative, in that there were no differences detected that could be attributed to an effect of time in the bioassay.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Sheetal Shah

PROVIDER: E-GEOD-27344 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:To investigate the systemic molecular changes occurring as a result of Dhr96 knockdown or over-expression, a comparison between knockdown or overexpression lines and their genetic controls were performed. 0-3 day old adult males or females were reared on 3 separate batches of diet (this was the standard diet we used for culturing Drosophila melanogaster and was made up of 10L water, 100g agar (USP #7060 Bio-serve), 350g Brewer’s dried yeast (Sunshine Health), 300g black treacle (Lyles), 150g sucrose (Tate & Lyle), 300g Difco dextrose (Becton Dickinson), 150g cornmeal (#1151, Bioserve), 100g wheatgerm (#1659, Bioserve), 200g soya bean flour (#S9633 Sigma Aldrich), 10g methyl-4-hydroxybenzoate (#H3647 Sigma Aldrich) in 10ml ethanol, 50ml proprionic acid (#P5561 Sigma Aldrich)). Each of these 3 batches was considered to represent independent biological replication. The RNA samples were hybridized to the Affymetrix Drosophila GeneChip 2. Previous Q-PCR analysis had indicated that there was differential Dhr96 gene expression in the knockdown or overexpression strains compared to their controls. (This was also inferred from phenotypes resulting from other distinct crosses.) However the first analysis carried out on these samples was to verify the levels of differential Dhr96 gene expression. From this analysis we found no evidence that Dhr96 is down-regulated in the knockdown line compared to its control for either Males or Females, as the confidence intervals for the fold change include the value of one and the p-values are large. Therefore the knockdown line and its control samples were excluded from further analysis. The overexpression line did however show significant up-regulation of Dhr96 expression relative to its control in both Males and Females, and was further analyzed. Flies of the appropriate genotype (Dhr96 knockdown or over-expression and their genetic controls) were reared on 3 different batches of diet (same recipe, prepared on different days). 0-3 day old adults were collected (males and females stored seperately) and each sample was hybridised independently to a chip.

Project description:Tsetse flies (Glossina spp.) are major vectors of African trypanosomes, causing either Human or Animal African Trypanosomiasis (HAT or AAT). Several approaches are developed to control the disease among which the anti-vector Sterile Insect Technique. Another approach in the frame of anti-vector strategies could consist in controlling the fly’s vector competence which needs identifying factors (genes, proteins, biological pathways, …) involved in this process. The present work aims to verify whether protein candidates identified under experimental controlled conditions on insectary-reared tsetse flies have their counterpart in field-collected flies. Glossina palpalis palpalis flies naturally infected with Trypanosoma congolense were sampled in two HAT/AAT foci in Southern Cameroon. After dissection, the proteome from guts of parasite-infected flies were compared to that from uninfected flies in order to identify quantitative and/or qualitative changes associated to infection. A total of 3291 proteins were identified of which 1818 could be quantified. The comparative analysis allowed identifying 175 proteins with significant decreased abundance in infected as compared to uninfected flies, while 61 proteins displayed increased abundance. Among the former are RNA binding proteins, kinases, actin, ribosomal proteins, endocytosis proteins, oxido-reductases, as well as proteins that are unusually found such as tsetse salivary proteins (Tsal) or Yolk proteins. Among the proteins with increased abundance are fructose-1,6-biphosphatase, serine proteases, membrane trafficking proteins, death proteins (or apoptosis proteins), and SERPINs (inhibitor of serine proteases, enzymes considered as trypanosome virulence factors) that displayed highest increased abundance. Sodalis, Wiggleswothia and Wolbachia proteins are strongly under-represented, particularly when compared to data from similar experimentation conducted under controlled conditions on T. brucei gambiense infected (or uninfected) G. palpalis gambiensis insectary reared flies. Comparing the overall recorded data, 364 proteins identified in gut extracts from field flies were shown to have a homologue in insectary flies. Discrepancies between the two studies may arise from differences in the species of studied flies and trypanosomes as well as in differences in environmental conditions in which the two experiments were carried out. Finally, the present study together with former proteomic and transcriptomic studies on the secretome of trypanosomes, on the gut extracts from insectary reared and on field collected tsetse flies, provide a pool of data and information on which to draw in order to perform further investigations on, for example, mammal host immunization or on fly vector competence modification via para-transgenic approaches.

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Expression data from transgenic Drosophila melanogaster adults which contain a knockdown effector of cyp6g1, compared to control flies

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