Bulked segregant analysis (BSA) is an efficient method to rapidly and efficiently map genes responsible for mutant phenotypes. This procedure, however, requires access to quantitative genetic markers that are polymorphic in the mapping population. We have developed a modification of BSA (BSR-Seq) that makes use of RNA-Seq reads to efficiently map genes even in populations for which no polymorphic markers have been previously identified. Because of the digital nature of next-generation sequencing (NGS) data, it is possible to conduct de novo SNP discovery and quantitatively genotype BSA samples using the same RNA-Seq data. In addition, analysis of the RNA-Seq data provides information on the effects of the mutant on global patterns of gene expression at no extra cost. In combination these results greatly simplify gene cloning experiments. To demonstrate the utility of this strategy BSR-Seq was used to clone the glossy3 (gl3) gene of maize. Mutants of the glossy loci exhibit altered accumulation of epicuticular waxes on juvenile leaves. We previously generated a large collection of glossy mutants using the Mu transposon system. By subjected a reference allele to BSR-Seq, we were able to map the gl3 locus to a ~2.3Mb interval that is consistent with the results of prior mapping experiments. The single gene located in the 2.3Mb mapping interval that contained a Mu insertion and whose expression was down-regulated in the mutant pool was subsequently demonstrated to be the gl3 gene via the analysis of multiple independently Mu transposon induced mutant alleles. The gl3 gene encodes a putative myb transcription factor, which directly or indirectly affects the expression of a number of genes involved in the biosynthesis of very-long-chain fatty acids.
Changes in genome content generated via segregation of non-allelic homologs.
No sample metadata fields
View SamplesTreatment of gonadectomized mice with estradiol, dihydrotestosterone or vehicle to compare gene expression in gastrocnemius.
Stimulation of both estrogen and androgen receptors maintains skeletal muscle mass in gonadectomized male mice but mainly via different pathways.
Sex, Specimen part, Disease, Compound
View SamplesWe examined the transcriptomes of murine "expandable hemangioblasts" (eHBs) and their blood and endothelial progeny, comparing them to the transcriptomes of murine embryonic stem (ES) cells, primary murine endothelial cells isolated from E11.5 yolk sacs or embryos, and E14.5 fetal liver hematopoietic stem cells. Overall design: Total RNAs were purified from lysates of cultured or primary cells, reverse transcribed, and sequenced on an Illumina HiSeq 2500.
An expandable, inducible hemangioblast state regulated by fibroblast growth factor.
No sample metadata fields
View SamplesMyotonic dystrophy type 1 (DM1) is an RNA dominant disease in which mutant transcripts containing an expanded CUG repeat (CUGexp) cause muscle dysfunction by interfering with biogenesis of other mRNAs. The toxic effects of mutant RNA are mediated partly through sequestration of splicing regulator Muscleblind-like 1 (Mbnl1), a protein that binds to CUGexp RNA. A gene that is prominently affected encodes chloride channel 1 (Clcn1), resulting in hyperexcitability of muscle (myotonia). To identify DM1-affected genes and study mechanisms for dysregulation, we performed global mRNA profiling in transgenic mice that express CUGexp RNA, as compared to Mbnl1 knockout and Clcn1 null mice. We found that the majority of changes induced by CUGexp RNA in skeletal muscle can be explained by reduced activity of Mbnl1, including many changes that are secondary to myotonia. The pathway most affected comprises genes involved in calcium signaling and homeostasis. Some effects of CUGexp RNA on gene expression are caused by abnormal alternative splicing or downregulation of Mbnl1-interacting mRNAs. However, several of the most highly dysregulated genes showed altered transcription, as indicated by parallel changes of the corresponding premRNAs. These results support the idea that trans-dominant effects of CUGexp RNA on gene expression in this transgenic model may occur at the level of transcription, RNA processing, and mRNA decay, and are mediated mainly but not entirely through sequestration of Mbnl1.
Transcriptional and post-transcriptional impact of toxic RNA in myotonic dystrophy.
Sex, Age
View SamplesAnalysis of collecting duct response to low NaCl or high NaCl diet at the gene expression level. Results provide insight into transcriptional changes in principal and intercalated cells that occur in response to changes in dietary NaCl. Overall design: Total RNA obtained from collecting duct cells isolated from mice fed low NaCl or high NaCl diet for 5 days.
Salt-sensitive transcriptome of isolated kidney distal tubule cells.
Sex, Specimen part, Cell line, Subject
View SamplesB cell chronic lymphocytic leukemia (CLL) is often preceded by a benign monoclonal or oligoclonal CD5+ B cell lymphocytosis. We have generated transgenic mice expressing a catalytically inactive, dominant-negative recombination activating gene 1 (dnRAG1 mice) in the periphery. These animals develop an early-onset indolent CD5+ B cell lymphocytosis, caused in part by a defect in secondary V(D)J rearrangements initiated to alter autoreactive B cell receptor specificity. Hypothesizing that the CD5+ B cells accumulating in dnRAG1 mice represent a CLL precursor, we crossed dnRAG1 mice with CLL-prone E-TCL1 mice to determine whether dnRAG1 expression in E-TCL1 mice accelerates the onset of CLL-like disease. We find that CD5+ B cell expansion and CLL progression occurs more rapidly and uniformly in double-transgenic mice (DTG mice) compared to E-TCL1 mice, but with similar phenotypic and leukemogenic features. To gain insight into genes or pathways responsible for CD5+ B cell accumulation in the transgenic mice, we performed comparative gene expression profiling studies using normal and CD5+ B cells isolated from wild-type and transgenic mice at either 12 weeks of age (pre-leukemia) or at CLL onset in DTG mice (using age-matched wild-type and single-transgenic mice as controls). These analyses confirm the upregulation of tolerogenic genes in CD5+ B cells and reveal a possible role for prolactin signaling in the regulation of receptor editing. This study suggests that a failure to remodel B cell antigen receptor genes in response to autoreactivity may promote the benign accumulation of CD5+ B cells, which may then be subjected to secondary genetic lesions that promote CLL progression.
Accelerated progression of chronic lymphocytic leukemia in Eμ-TCL1 mice expressing catalytically inactive RAG1.
Age, Specimen part
View SamplesAbstract from Vermillion et al: During vertebrate development, progenitor cells give rise to tissues and organs through a complex choreography that commences at gastrulation. A hallmark event of gastrulation is the formation of the primitive streak, a linear assembly of cells along the anterior-posterior (AP) axis of the developing organism. To examine the primitive streak at a single-cell resolution, we measured the transcriptomes of individual chick cells from the streak or the surrounding tissue (the rest of the area pellucida) in Hamburger-Hamilton stage 4 embryos. The single-cell transcriptomes were then ordered by the statistical method Wave-Crest to deduce both the relative position along the AP axis and the prospective lineage of single cells. The ordered transcriptomes reveal intricate patterns of gene expression along the primitive streak. Overall design: Examination of single-cells of stage 4 chicken embryos.
Spatial patterns of gene expression are unveiled in the chick primitive streak by ordering single-cell transcriptomes.
Specimen part, Subject
View SamplesWe examine how NGS sequencing of sperm can provide a window as to how particular perturbations of the sperm RNA profile from baseline may be indicative of male factor infertility, and may thus provide direction as to proper course of infertility treatment for couple. Overall design: NGS RNA-seq of 72 sperm samples from male partner of couples undergoing fertility treatment
Absence of sperm RNA elements correlates with idiopathic male infertility.
No sample metadata fields
View SamplesMyD88-independent signal transduction associated with Toll-like receptors (TLRs) 3 and TLR4 is mediated through the adapter protein TRIF (TIR-domain-containing adapter-inducing interferon-beta). It has been proposed that TLR signalling is important for the transcription of crucial inflammasome components like NLRP3, a process that has been termed "priming". In order to test whether TRIF signalling was required for the priming of inflammasome components, we performed a genome wide transcriptional analysis on wild-type and Trif-knockout bone marrow derived macrophages (BMMs) before and 1, 3 and 6 hours after phagocytosis of E. coli. These results indicated that TRIF was involved in the activation and not transcriptional priming of the NLRP3 inflammasome.
Detection of prokaryotic mRNA signifies microbial viability and promotes immunity.
Sex, Specimen part, Time
View SamplesThis SuperSeries is composed of the SubSeries listed below.
RNA expression profiling of human iPSC-derived cardiomyocytes in a cardiac hypertrophy model.
Specimen part
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