Project description:Prodynorphine-expressing neurons were targeted by eGFP-RPL10a, a transgene that is expressed under the control of the Pdyn promoter in a BAC containing the Pdyn backbone. Trangenic mice were made with FvB embryos via pro-nuclear injection. Transgene-positive progenies were bred to C57BL/6J for more than five generations before being used in the current experiment. Triplicates of 6-8 hypothalami were collected per sample from wild-type and ob/ob adult mice. Polysomal IP RNA and total Input RNA were purified from each sample.
Project description:Prodynorphine-expressing neurons were targeted by eGFP-RPL10a, a transgene that is expressed under the control of the Pdyn promoter in a BAC containing the Pdyn backbone. Trangenic mice were made with FvB embryos via pro-nuclear injection. Transgene-positive progenies were bred to C57BL/6J for more than five generations before being used in the current experiment. Triplicates of 6-8 hypothalami were collected per sample from wild-type and ob/ob adult mice. Polysomal IP RNA and total Input RNA were purified from each sample. Affymetrix GeneChip Mouse 430 2.0 arrays (900495) were used to identify transcripts that are specifically expressed in Pdyn neurons with a fold change (IP vs. Input) of >=2 in wild-type mice. The same microarrays were also used to identify Pdyn-specific transcripts that are differentially regulated transcriptionally by leptin deficiency with a fold change (ob IP vs. wt IP) of >=1.5.
Project description:TRAP translational profiling is a method that allows investigators to genetically characterize specific cell types in complex tissues such as mouse brain. Using this technique we obtained RNA-Seq data from actively translating transcripts present in neurons in the subthalamic nucleus of adult Cbln2-EGFP/Rpl10a (NIDA176) BAC transgenic mice.
Project description:BAC array purification of hippocampus astroglial ribosome-bound transcriptome in astrocytes from Aldh1l1:Rpl10a eGFP/ Cx43 KO and Aldh1l1:Rpl10a eGFP/ Cx43FL mice
Project description:Translating ribosome affinity purification (TRAP) was performed on spinal cord dissections pooled from 3-4 mice 21 days post birth that were positive for the eGFP-L10A fusion ribosomal marker protein under the expression of either the Chat promoter (Tg(Chat-EGFP/Rpl10a)DW167Htz) or the Snap25 promoter (Tg(Snap25-EGFP/Rpl10a)JD362Jdd). RNA-sequencing was performed on both TRAP and pre-immunoprecipitation (PreIP) control RNA samples.
Project description:The CCM endothelium is hypersensitive to angiogenesis and can induce a hypoxic program associated with changes in angiogenesis, inflammation, and endothelial-cell metabolism under normoxic conditions. However, the role of active drivers of angiogenesis as CCM disease modifiers in human disease remains unclear. To assess whether CCM reactive astrocytes with neuroinflammatory capacity respond to hypoxia-induced CCM exacerbation, we employed the astrocyte-specific (Aldh1l1-EGFP/Rpl10a) Translational Ribosome Affinity Purification (TRAP) system in combination with a CCM mouse model (Pdcd10BECKO;Aldh1l1-EGFP/Rpl10a). TRAP protocol was performed using Slco1c1-iCreERT2;Pdcd10fl/fl;Aldh1l1-EGFP/Rpl10a mice to isolate ribosomes from astrocytes as previously described. Astrocyte-TRAP mRNAs were from cerebral tissue of mice age P50 exposed to hypoxia or normoxia conditions.
Project description:We profiled the gene expression patterns of undisturbed endothelial cells in living animals using a novel ‘AngioTag’ zebrafish transgenic line that permits isolation of actively translating mRNAs from endothelial cells in their native environment. This transgenic line uses the endothelial cell-specific kdrl promoter to drive expression of an epitope tagged Rpl10a 60S ribosomal subunit protein, allowing for Translating Ribosome Affinity Purification (TRAP) of actively translating endothelial cell mRNAs. We also collected the whole embryo translatome using the TRAP protocol and a ubiquitously expressed tagged Rpl10a, and the endothelial transcritome by collecting the GFP+ endothelial cells that had the kdrl promoter driving GFP.
Project description:We profiled the gene expression patterns of undisturbed endothelial cells in living animals using a novel ‘AngioTag’ zebrafish transgenic line that permits isolation of actively translating mRNAs from endothelial cells in their native environment. This transgenic line uses the endothelial cell-specific kdrl promoter to drive expression of an epitope tagged Rpl10a 60S ribosomal subunit protein, allowing for Translating Ribosome Affinity Purification (TRAP) of actively translating endothelial cell mRNAs. We also collected the whole embryo translatome using the TRAP protocol and a ubiquitously expressed tagged Rpl10a, and the endothelial transcritome by collecting the GFP+ endothelial cells that had the kdrl promoter driving GFP.
Project description:TRAP translational profiling is a method that allows investigators to genetically characterize specific cell types in complex tissues such as mouse brain. Using this technique we obtained RNA-Seq data from actively translating transcripts present in neurons in the thalamus of adult Stard8-EGFP/Rpl10a (ES2342) mice.
Project description:TRAP translational profiling is a method that allows investigators to genetically characterize specific cell types in complex tissues such as mouse brain. Using this technique we obtained RNA-Seq data from actively translating transcripts present in neurons in the hypothalamus of adult Lhx5-EGFP/Rpl10a (DT154) mice.
