Project description:C57BL/6J (B6) and DBA/2J (D2) are two of the most commonly used inbred mouse strains in neuroscience research. However, the only currently available mouse genome is based entirely on the B6 strain sequence. Subsequently, oligonucleotide microarray probes are based solely on this B6 reference sequence, making their application for gene expression profiling comparisons across mouse strains dubious due to their allelic sequence differences, including single nucleotide polymorphisms (SNPs). The emergence of next-generation sequencing (NGS) and the RNA-Seq application provides a clear alternative to oligonucleotide arrays for detecting differential gene expression without the problems inherent to hybridization-based technologies. Using RNA-Seq, an average of 22 million short sequencing reads were generated per sample for 21 samples (10 B6 and 11 D2), and these reads were aligned to the mouse reference genome, allowing 16,183 Ensembl genes to be queried in striatum for both strains. To determine differential expression, 'digital mRNA counting' is applied based on reads that map to exons. The current study compares RNA-Seq (Illumina GA IIx) with two microarray platforms (Illumina MouseRef-8 v2.0 and Affymetrix MOE 430 2.0) to detect differential striatal gene expression between the B6 and D2 inbred mouse strains. We show that by using stringent data processing requirements differential expression as determined by RNA-Seq is concordant with both the Affymetrix and Illumina platforms in more instances than it is concordant with only a single platform, and that instances of discordance with respect to direction of fold change were rare. Finally, we show that additional information is gained from RNA-Seq compared to hybridization-based techniques as RNA-Seq detects more genes than either microarray platform. The majority of genes differentially expressed in RNA-Seq were only detected as present in RNA-Seq, which is important for studies with smaller effect sizes where the sensitivity of hybridization-based techniques could bias interpretation.

Project description:C57BL/6J (B6) and DBA/2J (D2) are two of the most commonly used inbred mouse strains in neuroscience research. However, the only currently available mouse genome is based entirely on the B6 strain sequence. Subsequently, oligonucleotide microarray probes are based solely on this B6 reference sequence, making their application for gene expression profiling comparisons across mouse strains dubious due to their allelic sequence differences, including single nucleotide polymorphisms (SNPs). The emergence of next-generation sequencing (NGS) and the RNA-Seq application provides a clear alternative to oligonucleotide arrays for detecting differential gene expression without the problems inherent to hybridization-based technologies. Using RNA-Seq, an average of 22 million short sequencing reads were generated per sample for 21 samples (10 B6 and 11 D2), and these reads were aligned to the mouse reference genome, allowing 16,183 Ensembl genes to be queried in striatum for both strains. To determine differential expression, 'digital mRNA counting' is applied based on reads that map to exons. The current study compares RNA-Seq (Illumina GA IIx) with two microarray platforms (Illumina MouseRef-8 v2.0 and Affymetrix MOE 430 2.0) to detect differential striatal gene expression between the B6 and D2 inbred mouse strains. We show that by using stringent data processing requirements differential expression as determined by RNA-Seq is concordant with both the Affymetrix and Illumina platforms in more instances than it is concordant with only a single platform, and that instances of discordance with respect to direction of fold change were rare. Finally, we show that additional information is gained from RNA-Seq compared to hybridization-based techniques as RNA-Seq detects more genes than either microarray platform. The majority of genes differentially expressed in RNA-Seq were only detected as present in RNA-Seq, which is important for studies with smaller effect sizes where the sensitivity of hybridization-based techniques could bias interpretation. There are 5 male and 5 female C57BL/6J animals and 5 male and 5 female DBA/2J. These are adult, naïve animals. One array is run for each animal for a total of 20 Mouse MOE430 2.0 arrays. The purpose is to determine baseline (naïve) gene expression differences in striatum for these two inbred strains. There are 12 male C57BL/6J animals and 12 male DBA/2J. These are adult, naïve animals. One array is run for each animal for a total of 24 Mouse Ref8 v2 arrays. The purpose is to determine baseline (naïve) gene expression differences in striatum for these two inbred strains. ***This submission represents the microarray component of the study

Project description:Prenatal alcohol exposure can result in fetal alcohol spectrum disorders (FASD). Not all women who consume alcohol during pregnancy have children with FASD and studies have shown that genetic factors can play a role in ethanol teratogenesis. We examined gene expression in embryos and placentae from C57BL/6J (B6) and DBA/2J (D2) mice following prenatal alcohol exposure. B6 fetuses are susceptible to morphological malformations following prenatal alcohol exposure while D2 are relatively resistant.Male and female B6 and D2 mice were mated for 2 hours in the morning, producing 4 embryonic genotypes: true-bred B6B6 and D2D2, and reciprocal B6D2 and D2B6. On gestational day 9, dams were intubated with 5.8 g/kg ethanol, an isocaloric amount of maltose dextrin, or nothing. Four hours later, dams were sacrificed and embryos and placentae were harvested. RNA was extracted, labeled and hybridized to Affymetrix Mouse Genome 430 v2 microarray chips. Data were normalized, subjected to analysis of variance and tested for enrichment of gene ontology molecular function and biological process using the Database for Annotation, Visualization and Integrated Discovery (DAVID).Several gene classes were differentially expressed in B6 and D2 regardless of treatment, including genes involved in polysaccharide binding and mitosis. Prenatal alcohol exposure altered expression of a subset of genes, including genes involved in methylation, chromatin remodeling, protein synthesis, and mRNA splicing. Very few genes were differentially expressed between maltose-exposed tissues and tissues that received nothing, so we combined these groups for comparisons with ethanol. While we observed many expression changes specific to B6 following prenatal alcohol exposure, none were specific for D2. Gene classes up- or down-regulated in B6 following prenatal alcohol exposure included genes involved in mRNA splicing, transcription, and translation.Our study identified several classes of genes with altered expression following prenatal alcohol exposure, including many specific for B6, a strain susceptible to ethanol teratogenesis. Lack of strain specific effects in D2 suggests there are few gene expression changes that confer resistance. Future studies will begin to analyze functional significance of the expression changes.

Project description:PurposeThe goal of the present study is to provide an independent assessment of the retinal transcriptome signatures of C57BL/6J (B6) and DBA/2J (D2) mice, and to enhance existing microarray data sets for accurately defining the allelic differences in the BXD recombinant inbred strains.MethodsRetinas from B6 and D2 mice (three of each) were used for the RNA sequencing (RNA-seq) analysis. Transcriptome features were examined for both strains. Differentially expressed genes between the two strains were identified, and bioinformatic analysis was performed to analyze the transcriptome differences between the B6 and D2 strains, including Gene Ontology (GO) analysis, Phenotype and Reactome enrichment, and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The RNA-seq data were then directly compared with one of the microarray data sets (Department of Defense [DoD] Retina Normal Affy MoGene 2.0 ST RMA Gene Level Microarray Database) hosted on GeneNetwork.ResultsRNA-seq provided an in-depth analysis of the transcriptome of the B6 and D2 retinas with a total of more than 30,000,000 reads per sample. More than 70% of the reads were uniquely mapped, resulting in a total of 18,100 gene counts for all six samples. A total of 1,665 genes were differentially expressed, with 858 of these more highly expressed in the B6 retinas and 807 more highly expressed in the D2 retinas. Several molecular pathways were differentially active between the two strains, including the retinoic acid metabolic process, endoplasmic reticulum lumen, extracellular matrix (ECM) organization, and the PI3K-Akt signaling pathway. The most enriched KEGG pathways were the pentose and glucuronate interconversions pathway, the cytochrome P450 pathway, the protein digestion and absorption pathway, and the ECM-receptor interaction pathway. Each of these pathways had a more than fourfold enrichment. The DoD Normal Retina Microarray Database provided expression profiling for 26,191 annotated transcripts for B6 mouse, D2 mouse, and 53 BXD strains. A total of 13,793 genes in this microarray data set were comparable to the RNA-seq data set. For the B6 and D2 retinas, the RNA-seq data and the microarray data were highly correlated with each other (Pearson's r=0.780 for the B6 mice and 0.784 for D2 mice). These results suggest that the microarray data set can reliably detect differentially expressed genes between the B6 and D2 retinas, with an overall accuracy of 91.1%. Examples of true positive and false positive genes are provided.ConclusionsRetinal transcriptome features of B6 and D2 mouse strains provide a useful reference for a better understanding of the mouse retina. Generally, the microarray database presented on GeneNetwork shows good agreement with the RNA-seq data, but we note that any allelic difference between B6 and D2 mice should be verified with the latter.

Project description:Previous reports have identified greater sensitivity to the locomotor-stimulating, sensitizing, and reinforcing effects of amphetamine in inbred C57BL/6J mice relative to inbred DBA/2J mice. The dopamine D3 receptor (D3R) plays an inhibitory role in the regulation of rodent locomotor activity, and exerts inhibitory opposition to D1 receptor (D1R)-mediated signaling. Based on these observations, we investigated D3R expression and D3R-mediated locomotor-inhibitory function, as well as D1R binding and D1R-mediated locomotor-stimulating function, in C57BL/6J and DBA/2J mice. C57BL/6J mice exhibited lower D3R binding density (-32%) in the ventral striatum (nucleus accumbens/islands of Calleja), lower D3R mRNA expression (-26%) in the substantia nigra/ventral tegmentum, and greater D3R mRNA expression (+40%) in the hippocampus, relative to DBA/2J mice. There were no strain differences in DR3 mRNA expression in the ventral striatum or prefrontal cortex, nor were there differences in D1R binding in the ventral striatum. Behaviorally, C57BL/6J mice were less sensitive to the locomotor-inhibitory effect of the D3R agonist PD128907 (10 microg/kg), and more sensitive to the locomotor-stimulating effects of novelty, amphetamine (1 mg/kg), and the D1R-like agonist +/- -1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8,-diol hydrochloride (SKF38393) (5-20 mg/kg) than DBA/2J mice. While the selective D3R antagonist N-(4-[4-{2,3-dichlorphenyl}-1 piperazinyl]butyl)-2-fluorenylcarboxamide (NGB 2904) (0.01-1.0 mg/kg) augmented novelty-, amphetamine-, and SKF38393-induced locomotor activity in DBA/2J mice, it reduced novelty-induced locomotor activity in C57BL/6J mice. Collectively, these results demonstrate that C57BL/6J mice exhibit less D3R-mediated inhibitory function relative to DBA/2J mice, and suggest that reduced D3R-mediated inhibitory function may contribute to heightened sensitivity to the locomotor-stimulating effects of amphetamine in the C57BL/6J mouse strain. Furthermore, these data demonstrate that comparisons between C57BL/6J and DBA/2J mouse strains provide a model for elucidating the molecular determinants of genetic influence on D3R function.

Dataset Information

Evaluating gene expression in C57BL/6J and DBA/2J mouse striatum using RNA-Seq and microarrays.

Publications

Evaluating gene expression in C57BL/6J and DBA/2J mouse striatum using RNA-Seq and microarrays.

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