ABSTRACT: gene expression is tested for an association with GSCs by comparing expression levels in ovary tips of c587-GAL4; hs-bam UAS-dpp females at 0 hr, 20 hr and 50 hr after heat shock ; results of two independent experiments are given as expt 1 and expt 2 Keywords: time course, heat shock, bam overexpression
Project description:Gene expression is tested for an association with GSCs by comparing expression levels in ovary tips of c587-GAL4; hs-bam UAS-dpp females at 0 hr, 20 hr and 50 hr after heat shock ; results of two independent experiments are given as expt 1 and expt 2 Experiment Overall Design: 0 hr ovary tips contain many GSCs Experiment Overall Design: 20 hr hs-bam has induced differentiation of all GSCs Experiment Overall Design: 50 hr Bam has turned over and cystocytes have reverted to become GSCs again Experiment Overall Design: see Kai et al. (2005) Dev. Biol, in press.
Project description:This series compares gene expression between germ line stem cells (GSCs) purified either from bam mutant or Dpp-overexpressing ovaries, with gene expression in Kc cells Keywords: bam mutant, Dpp overexpression
Project description:To determine the extent of alternative 3' end cleavage during Drosophila spermatogenesis, we performed 3' end sequencing of Drosophila testes from flies at different time points post heat shock (pHS) that were mutant for bam and also had a heat shock inducible transgene expressing Bam. We then performed Polysome profiling of testes at 24, 48 and 72 hours PHS and created 3' end sequencing libraries from the free, 40S, 60s, 80S, 2-3 ribosomes, and 4+ ribosomes fractions for each dataset
Project description:This series compares gene expression between germ line stem cells (GSCs) purified either from bam mutant or Dpp-overexpressing ovaries, with gene expression in Kc cells Experiment Overall Design: 3 replicates of GSCs from bam mutant ovaries Experiment Overall Design: 3 replicates of GSCs from Dpp overexpressing ovaries Experiment Overall Design: 3 replicates of Kc tissue culture cells
Project description:A 2-hour heat-shock at 37C was used to activate hs-FLP and an actin5C-FRT-stop-FRT-GAL4 transgene in larvae carrying any possible combination of the genetic elements UAS-Myc, UAS-Atu-IR, Max-/-. 48 hours later 16-27 wing imaginal discs were isolated from wandering L3 larvae and polyA-RNA was processed for sequencing.
Project description:To investigate the role of CPES in germ cell differentiation during spermatogenesis in Drosophila testis. We have generated cpes null mutants using ends-out homologus recombination and rescued with Bam-Gal4 and UAS-CPES in cpes mutant background. We then dissected 200 pairs of testes for each of the 3 replicates from wild type, cpes mutant and Rescue (Bam-Gal4 and UAS-CPES) Drosophila males and total RNA was extracted using Trizol and RNA-Clean and concentrater column. About 1ug of RNA in 20ul was submitted for bulk RNAseq using Illumina machine (HWI-ST1276).
Project description:HUVEC (Human Umbilical Vein Endothelial Cells) cells were exposed to heat shock (42 degrees Celcius) for different time points (1hr, 3 hrs, 6 hr and 12 hrs) and compared to non heat-shock cells. External normalization controls were added in equal amounts to equivalent amounts of total RNA.
Project description:Cryptorchidism and scrotal heating result in abnormal spermatogenesis but the mechanism(s) proscribing this temperature sensitivity are unknown. It was previously reported that the AKR/N or MRL/MpJ-+/+ mouse testis is more heat resistant than the testis from the C57BL/6 strain. We have attempted to probe into the mechanism(s) involved in heat sensitivity by examining global gene expression profiles of normal and heat-treated testes from C57BL/6, AKR/N and MRL/MpJ-+/+ mice by microarray analysis. In the normal C57BL/6 testis, 415 and 416 transcripts were differentially expressed (at least two-fold higher or lower) when compared to the normal AKR/N and MRL/MpJ-+/+ testis, respectively. The AKR/N and MRL/MpJ-+/+ strains revealed 268 differentially expressed transcripts between them. There were 231 transcripts differentially expressed between C57BL/6 and two purported heat-resistant strains, AKR/N and MRL/MpJ-+/+. Next, the testes of C57BL/6 and AKR/N mice were exposed to 43ºC for 15 min and harvested at different time points for TUNEL studies and microarrays. An increase of TUNEL-positive germ cell numbers was significant 8 hr after heat exposure in the C57BL/6 mouse. However, this increase was not observed in the AKR/N mouse until 10 hr after heat exposure. All tubules showed germ cell loss and disruption in C57BL/6 testis 24 hr after heat shock. In contrast, although a number of seminiferous tubules showed an abnormal morphology 24 hr post-heat shock in the AKR/N mouse, many tubules still retained a normal structure. Numerous transcripts exhibited differential regulation between the two strains within 24 hours after heat exposure. The differentially expressed transcripts in the testes 8 hr after heat exposure were targeted to identify the genes involved in the initial response rather than those due to germ cell loss. Twenty transcripts were significantly down-regulated and 19 genes were up-regulated by hyperthermia in C57BL/6 and did not show a parallel change in the AKR/N testis. Conversely, heat shock resulted in 30 up-regulated transcripts and 31 down-regulated transcripts in AKR/N that were not similarly regulated in C57BL/6. A number of genes shared similar differential expression patterns and differential regulation by hyperthermia in both strains of mice. Taken together, the present study indicates the diverse genetic backgrounds in the three strains lead to major differences in normal testis gene expression profiles while the differences in heat shock responses involves a significantly smaller number of genes. The data generated may provide insights regarding gene networks and pathways involved in heat stress and their relationship to spermatogenesis.
Project description:Fly strains: All transgenes are P[+] in w strains. w+;+;Act 5c > CD2 > GAL4 UAS-GFP (Neufeld et al. 1998 ); y w hs-FLP122; +; UAS-dMyc (Zaffran et al. 1998 ). y w hs-FLP122; +; +. Adult flies and larvae were raised in regular fly food consisting of cornmeal and molasses at 25°C. Larvae overexpressing either UAS-regulated dMyc;GFP or GFP alone transgenes were generated using the Flp/Gal4 method (Struhl and Basler 1993 ; Pignoni and Zipursky 1997 ; Neufeld et al. 1998 ). Larvae were staged from hatching and raised at a density of 50 per vial at 25°C. Third instar larvae (110 h after egg deposition, AED) were heat shocked at 37°C for 2 h, and larvae were collected 7 h after heat shock (~120 h AED). Total RNA was isolated using TRIzol reagent (Invitrogen) as described by manufacturer followed by RNeasy (Qiagen) clear up. cRNA targets were generated using a standard amino-allyl labeling protocol, where 30 µg each of "experimental" (dMyc;GFP: hs-FLP122; Act-GAL4, UAS-GFP; UAS-dMyc) and "reference" (GFP only :ywhs-FLP122; Act-GAL4, UAS-GFP; +) total RNAs were coupled to either Cy3 or Cy5 fluorophores. Paired labeled targets were processed on microarrays using protocols described elsewhere (Fazzio et al. 2001 ). Posthybridized arrays were scanned using a GenePix 4000 scanner (Axon Instruments). Data were generated from five independent replicates (two with one dye orientation and three with the reversed dye orientation) at 7h and 14h Keywords: repeat sample