Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Chromatin immunopreciptation of mouse overexpressing ELAV-like protein HuD

ABSTRACT: Post-transcriptional mechanisms play an important role in the control of gene expression. RNA-binding proteins are key players in the post-transcriptional control of many neural genes and they participate in multiple processes, from RNA splicing and mRNA transport to mRNA stability and translation. Our laboratory has developed the first mouse model overexpressing a RNA-binding protein, the ELAV-like protein HuD, in the CNS under the control of the CaMKinII alpha promoter. Initial behavioral characterization of the mice revealed that they had significant learning deficits together with abnormalities in prepulse inhibition (PPI). At the molecular level, we found that the expression of the growth-associated protein GAP-43, one of the targets of HuD, was increased in the hippocampus of HuD transgenic mice. Besides binding and stabilizing the GAP-43 mRNA, HuD was shown to bind in vitro or in vivo the mRNAs of acetylcholinesterase, tau, p21, neuroserpin, and MARCKS among others. To identify additional targets of HuD, we propose to pull down the RNAs bound to myc-tagged HuD in vivo in the brains of HuD transgenic mice, to isolate these bound RNAs and use these to probe DNA microarrays. We will use pull downs using non-immune IgGs as controls. To test our hypothesis we propose 2 specific aims:; 1) To identify the targets of HuD in HuD overexpressor mice and; 2) To compare these target mRNAs to those we identified previously as having increased levels of expression in the hippocampus of HuD transgenic mice (see protocol # Perrone-Bizzozero-5R01NS030255-12) and/or those that show increased expression in dentate granule cells of HuD transgenic mice ( protocol # perro-affy-mouse-309741); Based upon the role of the RNA-binding HuD in neuronal development and synaptic plasticity, we expect that HuD targets will include mRNAs for proteins involved in these processes. All mice are in C57BL/6 background and are male approximately 60 days old. To identify target of HuD in our transgenic mice, we will homogenize the homogenize the hippocampi (2 per animal) of 3 transgenic mice and use these protein extracts for immunoprecipitation assays. Briefly, transgenic myc-tagged HuD protein will be immunoprecipitated using myc-tag antibodies and protein-G agarose beads and samples will be sent to T-Gen for RNA isolation, single round amplification and probing of DNA microarrays. We will use non-immune IgG as a negative control. Conditions:; Pooled Extracts from 3 transgenic mice IP with myc-tag antibodies: Triplicates; Pooled extract from 3 transgenic mice IP with non-immune IgGs (negative control): duplicates

ORGANISM(S): Mus musculus

SUBMITTER: Elizabeth Salomon

PROVIDER: E-GEOD-11333 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Post-transcriptional mechanisms play an important role in the control of gene expression. RNA-binding proteins are key players in the post-transcriptional control of many neural genes and they participate in multiple processes, from RNA splicing and mRNA transport to mRNA stability and translation. Our laboratory has developed the first mouse model overexpressing a RNA-binding protein, the ELAV-like protein HuD, in the CNS under the control of the CaMKinII alpha promoter. Initial behavioral characterization of the mice revealed that they had significant learning deficits together with abnormalities in prepulse inhibition (PPI). At the molecular level, we found that the expression of the growth-associated protein GAP-43, one of the targets of HuD, was increased in the hippocampus of HuD transgenic mice. Besides binding and stabilizing the GAP-43 mRNA, HuD was shown to bind in vitro or in vivo the mRNAs of acetylcholinesterase, tau, p21, neuroserpin, and MARCKS among others. To identify additional targets of HuD, we propose to pull down the RNAs bound to myc-tagged HuD in vivo in the brains of HuD transgenic mice, to isolate these bound RNAs and use these to probe DNA microarrays. We will use pull downs using non-immune IgGs as controls. To test our hypothesis we propose 2 specific aims: 1) To identify the targets of HuD in HuD overexpressor mice and 2) To compare these target mRNAs to those we identified previously as having increased levels of expression in the hippocampus of HuD transgenic mice (see protocol # Perrone-Bizzozero-5R01NS030255-12) and/or those that show increased expression in dentate granule cells of HuD transgenic mice ( protocol # perro-affy-mouse-309741) Based upon the role of the RNA-binding HuD in neuronal development and synaptic plasticity, we expect that HuD targets will include mRNAs for proteins involved in these processes. All mice are in C57BL/6 background and are male approximately 60 days old. To identify target of HuD in our transgenic mice, we will homogenize the homogenize the hippocampi (2 per animal) of 3 transgenic mice and use these protein extracts for immunoprecipitation assays. Briefly, transgenic myc-tagged HuD protein will be immunoprecipitated using myc-tag antibodies and protein-G agarose beads and samples will be sent to T-Gen for RNA isolation, single round amplification and probing of DNA microarrays. We will use non-immune IgG as a negative control. Conditions: Pooled Extracts from 3 transgenic mice IP with myc-tag antibodies: Triplicates Pooled extract from 3 transgenic mice IP with non-immune IgGs (negative control): duplicates Keywords: dose response

Project description:Post-transcriptional mechanisms play an important role in the control of gene expression. RNA-binding proteins are key players in the post-transcriptional control of many neural genes and they participate in multiple processes, from RNA splicing and mRNA transport to mRNA stability and translation. Our laboratory has developed the first mouse model overexpressing a RNA-binding protein, the ELAV-like protein HuD, in the CNS under the control of the CaMKinII alpha promoter. Initial behavioral characterization of the mice revealed that they had significant learning deficits together with abnormalities in prepulse inhibition (PPI). At the molecular level, we found that the expression of the growth-associated protein GAP-43, one of the targets of HuD, was increased in the hippocampus of HuD transgenic mice. To characterize these mice further and to evaluate the utility of these animals in understanding human diseases, we propose to use DNA microarray methods. To characterize the pattern of gene expression in the hippocampus of HuD overexpressor mice; Based on the behavioral and molecular properties of our HuD transgenic mice we hypothesize that these animals may be good models for the studying the basis of learning disabilities and of diseases that show deficits in PPI such as fetal alcohol syndrome and schizophrenia. All mice are in C57BL/6 background and are male approximately 30 days old. Animals are bred and sacrificed according to our approved animal protocol. Brains will be rapidly dissected on ice, quickly frozen and stored at -80C until analysis. Frozen brains will be embedded in OCT and shipped to the DNA microarray facility at Duke University where LCM was performed. Dentate granule cells from control and HuD transgenic mice will be isolated by Susan Su, who will also perform RNA isolation and the first round of RNA amplification. For our first experiment, we want to examine the pattern of gene expression in the hippocampus of 2 transgenic mice and 2 non-transgenic littermates.

Project description:Post-transcriptional mechanisms play an important role in the control of gene expression. RNA-binding proteins are key players in the post-transcriptional control of many neural genes and they participate in multiple processes, from RNA splicing and mRNA transport to mRNA stability and translation. Our laboratory has developed the first mouse model overexpressing a RNA-binding protein, the ELAV-like protein HuD, in the CNS under the control of the CaMKinII alpha promoter. Initial behavioral characterization of the mice revealed that they had significant learning deficits together with abnormalities in prepulse inhibition (PPI). At the molecular level, we found that the expression of the growth-associated protein GAP-43, one of the targets of HuD, was increased in the hippocampus of HuD transgenic mice. To characterize these mice further and to evaluate the utility of these animals in understanding human diseases, we propose to use DNA microarray methods. To characterize the pattern of gene expression in the hippocampus of HuD overexpressor mice Based on the behavioral and molecular properties of our HuD transgenic mice we hypothesize that these animals may be good models for the studying the basis of learning disabilities and of diseases that show deficits in PPI such as fetal alcohol syndrome and schizophrenia. All mice are in C57BL/6 background and are male approximately 30 days old. Animals are bred and sacrificed according to our approved animal protocol. Brains will be rapidly dissected on ice, quickly frozen and stored at -80C until analysis. Frozen brains will be embedded in OCT and shipped to the DNA microarray facility at Duke University where LCM was performed. Dentate granule cells from control and HuD transgenic mice will be isolated by Susan Su, who will also perform RNA isolation and the first round of RNA amplification. For our first experiment, we want to examine the pattern of gene expression in the hippocampus of 2 transgenic mice and 2 non-transgenic littermates. Keywords: HuD, hippocampus

Project description:Post-transcriptional mechanisms play an important role in the control of gene expression. RNA-binding proteins are key players in the post-transcriptional control of many neural genes and they participate in multiple processes, from RNA splicing and mRNA transport to mRNA stability and translation. Our laboratory has developed the first mouse model overexpressing a RNA-binding protein, the ELAV-like protein HuD, in the CNS under the control of the CaMKinII alpha promoter. Initial behavioral characterization of the mice revealed that they had significant learning deficits together with abnormalities in prepulse inhibition (PPI). At the molecular level, we found that the expression of the growth-associated protein GAP-43, one of the targets of HuD, was increased in the hippocampus of HuD transgenic mice. To characterize these mice further and to evaluate the utility of these animals in understanding human diseases, we propose to use DNA microarray methods. To test our hypothesis we propose 2 specific aims: 1)To characterize the pattern of gene expression in the hippocampus of HuD overexpressor mice 2)To compare the pattern of gene expression in our mouse model with that in the hippocampus of rats prenatally exposed to alcohol (FAS model) and in post-mortem tissues of patients with schizophrenia; Based on the behavioral and molecular properties of our HuD transgenic mice we hypothesize that these animals may be good models for the studying the basis of learning disabilities and of diseases that show deficits in PPI such as fetal alcohol syndrome and schizophrenia. All mice are in C57BL/6 background and are male approximately 60 days old. Initial studies were performed in animals that were not subjected to any experimental manipulation. Animals were bred and sacrificed according to our approved animal protocol. The brain was rapidly dissected on ice and we isolated the hippocampus, which has the highest expression of the transgene. After dissection both hippocampi were frozen in liquid nitrogen, pooled and stored at -80°C until analysis. RNA samples were isolated using RNAeasy Qiagen columns. For our first experiment, we want to examine the pattern of gene expression in the hippocampus of 3 transgenic mice and 3 non-transgenic littermates. RNAs from the 6 hippocampi were of high quality as revealed by the integrity of the 28S and 18S rRNA. We will provide 6 samples containing 10 ug of RNA each in DEPC water at a concentration of about 0.5 ug/ul. Three of the samples (#1, #2 and #3) are from transgenic mice and three from their non-transgenic littermates (#4, #5 and #6).

Project description:Our laboratory has developed the first mouse model overexpressing a RNA-binding protein, the ELAV-like protein HuD, in the CNS under the control of the CaMKinII alpha promoter. Initial behavioral characterization of the mice revealed that they had significant learning deficits together with abnormalities in prepulse inhibition (PPI). At the molecular level, we found that the expression of the growth-associated protein GAP-43, one of the targets of HuD, was increased in the hippocampus of HuD transgenic mice. To characterize these mice further and to evaluate the utility of these animals in understanding human diseases, we propose to use DNA microarray methods. To test our hypothesis we propose 3 specific aims: 1) To characterize the pattern of gene expression in the hippocampus of HuD overexpressor mice 2) To compare the pattern of gene expression in our mouse model with that in the hippocampus of rats prenatally exposed to alcohol (FAS model) and 3) To compare the pattern of gene expression in our mouse model with that shown in post-mortem tissues of patients with schizophrenia. In our previous protocols we examined the pattern of gene expression in our HuD transgenic mice and in rats prenaltally exposed to alcohol. A report by another group (Hakak et al, 2001) showed that three of the HuD targets were upregulated in the prefrontal cortex of patients with schizophrenia. To evaluate whether other target of HuD may be affected in this illness, in the current protocol, we want to compare the pattern of expression in our transgenic mice with in tissue from patients with schizophrenia Based on the behavioral and molecular properties of our HuD transgenic mice we hypothesize that these animals may be good models for the studying the basis of learning disabilities and of diseases that show deficits in PPI such as fetal alcohol syndrome and schizophrenia. All 28 samples are derived from cerebellar tissues for patients with schizophrenia and matched controls. The specimens were obtained from the Maryland Brain Collection according to NIH guidelines for confidentially and privacy. The protocol used in these studies was reviewed by our HRRC which found that our studies do not fall within the category of protocols monitored by the IRB (see attached letter form the HRRC). Specimens from 14 patients with a diagnosis of schizophrenia performed according to DSM-IV criteria and 14 sex-, age- and PMI-matched controls was included in this study. No differences were found between patients and control subjects in the average age (45±12 versus 43±10 years, p=0.86) or PMI (12±5 versus 16±6 hours, p=0.11). We will provide 28 samples containing 5 ug of RNA each in DEPC water (see validation of the quality of the RNA below). In addition, we include in our study animals treated with haloperidol as control for medication. These samples will be submitted in a separate protocol. Keywords: other

Project description:Post-transcriptional mechanisms play an important role in the control of gene expression. RNA-binding proteins are key players in the post-transcriptional control of many neural genes and they participate in multiple processes, from RNA splicing and mRNA transport to mRNA stability and translation. Our laboratory has developed the first mouse model overexpressing a RNA-binding protein, the ELAV-like protein HuD, in the CNS under the control of the CaMKinII alpha promoter. Initial behavioral characterization of the mice revealed that they had significant learning deficits together with abnormalities in prepulse inhibition (PPI). At the molecular level, we found that the expression of the growth-associated protein GAP-43, one of the targets of HuD, was increased in the hippocampus of HuD transgenic mice. To characterize these mice further and to evaluate the utility of these animals in understanding human diseases, we propose to use DNA microarray methods. To test our hypothesis we propose 2 specific aims: 1)To characterize the pattern of gene expression in the hippocampus of HuD overexpressor mice 2)To compare the pattern of gene expression in our mouse model with that in the hippocampus of rats prenatally exposed to alcohol (FAS model) and in post-mortem tissues of patients with schizophrenia Based on the behavioral and molecular properties of our HuD transgenic mice we hypothesize that these animals may be good models for the studying the basis of learning disabilities and of diseases that show deficits in PPI such as fetal alcohol syndrome and schizophrenia. All mice are in C57BL/6 background and are male approximately 60 days old. Initial studies were performed in animals that were not subjected to any experimental manipulation. Animals were bred and sacrificed according to our approved animal protocol. The brain was rapidly dissected on ice and we isolated the hippocampus, which has the highest expression of the transgene. After dissection both hippocampi were frozen in liquid nitrogen, pooled and stored at -80C until analysis. RNA samples were isolated using RNAeasy Qiagen columns. For our first experiment, we want to examine the pattern of gene expression in the hippocampus of 3 transgenic mice and 3 non-transgenic littermates. RNAs from the 6 hippocampi were of high quality as revealed by the integrity of the 28S and 18S rRNA. We will provide 6 samples containing 10 ug of RNA each in DEPC water at a concentration of about 0.5 ug/ul. Three of the samples (#1, #2 and # 3) are from transgenic mice and three from their non-transgenic littermates (#4, #5 and #6). Keywords: other

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Chromatin immunopreciptation of mouse overexpressing ELAV-like protein HuD

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