We developed a mouse line targeting midbrain dopamine neurons for Translating Ribosome Affinity Purification (TRAP). Here, we briefly report on the basic characterization of this mouse line including confirmation of expression of the transgene in midbrain dopamine neurons and validation of its effectiveness in capturing mRNA from these cells. We also report a translational profile of these neurons which may be of use to investigators studying the gene expression of these cells. Finally, we have donated the line to Jackson Laboratories for distribution and use in future studies.
Generation and characterization of a mouse line for monitoring translation in dopaminergic neurons.
Sex, Specimen partView Samples
Comparative analysis can provide important insights into complex biological systems. As demonstrated in the accompanying paper, Translating Ribosome Affinity Purification (TRAP), permits comprehensive studies of translated mRNAs in genetically defined cell populations following physiological perturbations.
Application of a translational profiling approach for the comparative analysis of CNS cell types.
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Three cell types, intermediolateral column motoneurons, medial motoneurons, and lateral motoneurons were isolated from a single adult spinal cord using laser capture microscopy. Four hundred captures were collected for each cell type. For a given cell type, RNA was extracted from the 400 captures using an Arcturus picopure kit. RNA was split in half and two targets were produced using a double amplification protocol. Each target was hybridized to Affymetrix chips and signals were normalized with R-pack. Inverse logs are provided. Five animals were used in these experiments, and all three cell types were collected from each animal. Thus, for each cell type, there are five biological replicates, and for each biological replicate there are two technical replicates. In all thirty chips were analyzed. Techinical replicates are indicated as Set 1 and Set 2. Animal numbers are indicated by Pair1 through Pair 5.
Divergence between motoneurons: gene expression profiling provides a molecular characterization of functionally discrete somatic and autonomic motoneurons.
Specimen partView Samples
SynapTRAP. Identification of Synaptic mRNA of neurons of the cortex. Technique combines sucrose percoll fractionation of a synaptically rich sample (SN) and TRAP tagged ribosome IP (PreIP and PostIP). This experiment uses pan neuronal SNAP25 mice and a cortical dissection. Overall design: Three replicates of four samples.
Transcriptomic Analysis of Ribosome-Bound mRNA in Cortical Neurites <i>In Vivo</i>.
Specimen part, SubjectView Samples
We report the mRNAs bound to ribosomes in peripheral astrocyte processes, thus suggesting local translation in astrocytes. Overall design: Isolation of a synaptoneurosome fraction from mouse cortex in which Astrocyte ribosomes were labeled with eGFP. Immunoprecipitation of GFP from this fraction to obtain astrocyte ribosomes away from the cell body. Performed RNA seq on these purified ribosomes and their bound mRNAs.
Astrocytes locally translate transcripts in their peripheral processes.
Specimen part, Cell line, SubjectView Samples
The discovery of genetic variants in the CHRNA5-CHRNA3-CHRNB4 gene cluster associated with heavy smoking and higher relapse risk has led to the identification of the midbrain habenula- interpeduncular axis as a critical relay circuit in the control of nicotine addiction
Reexposure to nicotine during withdrawal increases the pacemaking activity of cholinergic habenular neurons.
Specimen part, DiseaseView Samples
Background: Recent characterization of the transcriptional landscape of cell lines and whole tissues has suggested widespread transcription of the genome, including loci that produce regulatory non-coding RNAs that function within the nucleus. Methods: Here, we have defined the nuclear transcriptional landscape of the three major cellular divisions of the nervous system using flow sorting of genetically labeled nuclei from bacTRAP mouse lines followed by characterization the unique expression of coding, non-coding and intergenic RNAs in the mature mouse brain with RNAseq, and validation with independent methods. Results: Our findings reveal diverse expression across the cell-types of all classes of RNAs, including long non-coding RNAs - several of which were confirmed as highly enriched in the nuclei of specific cell-types using anatomical methods. Finally, we also discovered several examples of cell-type specific expression of tandem gene fusions, and report the first cell-type specific expression of circular RNAs, notably a neuron specific and nuclear enriched RNA arising from the gene Hnrnpu. Conclusion: These non-coding RNA expression data should provide an important context for studies evaluating the function of a variety of ncRNA in the nervous system. Overall design: Three to four independent replicate samples (each from one mouse) were collected for each of three sample types: Neuronal nuclear RNA, Astrocyte nuclear RNA, Oligodendrocyte nuclear RNA. Controls include low-coverage presorted nuclear RNA from each mouse.
A Comprehensive Analysis of Cell Type-Specific Nuclear RNA From Neurons and Glia of the Brain.
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The mature CNS contains PDGFRA+ oligodendrocyte progenitor cells (OPC) which may remain quiescent, proliferate, or differentiate into oligodendrocytes. In human gliomas, rapidly proliferating Olig2+ cells resembling OPCs are frequently observed. We sought to identify, in vivo, candidate pathways uniquely required for OPC differentiation or quiescence. Using the bacTRAP methodology, we generated and analyzed mouse lines for translational profiling the major cells types (including OPCs), in the normal mouse brain. We then profiled oligodendoglial (Olig2+) cells from a mouse model of Pdgf-driven glioma. This analysis confirmed that Olig2+ tumor cells are most similar to OPCs, yet, it identified differences in key progenitor genes - candidates for promotion of differentiation or quiescence.
Candidate pathways for promoting differentiation or quiescence of oligodendrocyte progenitor-like cells in glioma.
Specimen partView Samples
Comparison of polysomal profiles of murine adult olig2 cortical progenitors, murine tumor olig2 cells derived from hPDGF-B-driven glioblastomas, and murine olig2 proliferative recruited glioma cells contributing to the tumor mass but not derived from the cell of origin
Recruited cells can become transformed and overtake PDGF-induced murine gliomas in vivo during tumor progression.
Specimen partView Samples