Temporal- and Orbital Shear Stress-Dependent Changes in Opossum Kidney (OK) Cell Transcription
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PROVIDER: PRJNA649290 | ENA |
REPOSITORIES: ENA
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Project description:We previously demonstrated that OK cells cultured under orbital shear stress have morphological and functional features that more closely resemble the proximal tubule in vivo. This study was designed to identify progressive transcriptional changes with increasing exposure time to shear stress and to compare the transcriptome of cells cultured under shear stress vs static conditions.
2020-09-29 | GSE155315 | GEO
Project description:The OK cell line derived from kidney of a female opossum Didelphys virginiana has proven to be a useful model in which to investigate the unique regulation of ion transport and membrane trafficking mechanisms in the proximal tubule (PT). Sequence data and comparison of the transcriptome of this cell line to eutherian mammal PTs would further broaden the utility of this culture model. However, genomic sequence for Didelphys virginiana is not available and although a draft genome sequence for the opossum Monodelphis domestica (sequenced in 2012 by the Broad Institute) exists, its relatedness and similarity of the transcriptome to the Didelphys virginiana species is not known. The Monodelphis domestica sequence is not highly annotated, and the majority of transcripts are predicted rather than experimentally validated. Using deep RNA sequencing of the Didelphys virginiana OK cell line we characterized its transcriptome using de novo transcriptome assembly and alignment to the Monodelphis domestica genome. The quality of the de novo assembled transcriptome was assessed by the extent of homology to sequences in nucleotide and protein databases. Gene expression levels in the OK cell line, from both the de novo transcriptome and genes aligned to the Monodelphis domestica genome, were compared to publicly available rat kidney nephron segment expression data. Our studies demonstrate the expression in OK cells of numerous PT specific ion transporters and other key proteins relevant for rodent and human PT function. The sequence and expression data reported here provide a new and important resource for studies on the regulation of PT mRNA and protein expression.
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Project description:Array experiment: Animals were divided into a control group (naive), which did not undergo any experimental manipulation (n=20) and two experimental groups, one receiving an intra-orbital nerve transection (IONT, n=60) and the other an intra-orbital nerve crush (IONC, n=60) Sterile precautions were maintained for all surgical procedures. The left ON was intraorbitally axotomized according to procedures that are standard in our laboratory. (REF) Briefly, an incision was made in the superior orbital rim, the superoexternal orbital contents were dissected, and the superior and external rectus muscles were removed. When performing IONT, the dura mater was opened longitudinally to spare the blood supply, and the optic nerve was transected 0.5 mm from the optic disc. To perform IONC the optic nerve was crushed 3 mm from the optic disc during 10 seconds using a pair of watchmakers forceps (Vidal-Sanz et al., 1988, 1991; Villegas-Perez et al., 1993). Before and after the procedure, the eye fundus was observed through the operating microscope to assess the integrity of the retinal blood flow. Tissue processing and RNA extraction: Animals were kept the appropriate time post injury (12h, 24h, 48h, 3d 7d and 15d, n=8-12 per time point) and then sacrificed by an intraperitoneal overdose of sodium pentobarbital. Left retinas were fresh dissected and immediately frozen in liquid nitrogen. Four retinas from each time point, animal group and biological replica were pooled and RNA was extracted using Trizol (Invitrogen). RNA was further cleaned through RNAeasy mini kit columns (Quiagen). The RNA integrity was checked using a Bioanalyzer (Agilent Technologies, USA) and concentrations were determined using a NanoDrop (NanoDrop Technologies, Wilmington, DE, USA).
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Project description:Porcine endothelial cells were preconditioned by a basal level shear stress of 15 ± 15 dynes/cm2 at 1 Hz for 24 hours, and an acute increase in shear stress frequency (2 Hz) was then applied. The transcriptomics studies using microarray identified genes that were sensitive to the elevated shear frequency. keywords: adaptation, shear stress, frequency, microarray, gene expression Porcine endothelial cells were preconditioned by a basal level shear stress of 15 ± 15 dynes/cm2 at 1 Hz for 24 hours, and an acute increase in shear stress magnitude (2 Hz) was then applied. Gene expression at multiple time points was measured using microarray.
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