Project description:We created mice, which are deficient for Myc specifically in cardiac myocytes by crossing crossed Myc-floxed mice (Mycfl/fl) and MLC-2VCre/+ mice. Serial analysis of earlier stages of gestation revealed that Myc-deficient mice died prematurely at E13.5-14.5. Morphological analyses of E13.5 Myc-null embryos showed normal ventricular size and structure; however, decreased cardiac myocyte proliferation and increased apoptosis was observed. BrdU incorporation rates were also decreased significantly in Myc-null myocardium. Myc-null mice displayed a 3.67-fold increase in apoptotic cardiomyocytes by TUNEL assay. We examined global gene expression using oligonucleotide microarrays. Numerous genes involved in mitochondrial death pathways were dysregulated including Bnip3L and Birc2. Keywords: wildtype vs Myc-null
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare the difference of NGS-derived transcriptome profiling between DN thymocytes from Toxf/f and Toxf/fPdcd1Cre mice. Methods: mRNA profiles of DN thymocytes sorted by FACS from 3-4-week-old Toxf/f and Toxf/fPdcd1Cre mice were generated by deep sequencing, in triplicate, using Illumina Novaseq6000. The sequence reads that passed quality filters were analyzed at the transcript level with qRT–PCR using TaqMan and SYBR Green assays. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (Build Ensembl_release100) and identified 19,045 transcripts in the DN thymocytes of Toxf/f and Toxf/fPdcd1Cre mice with BWA workflow. RNA-seq data showed approximately 1.4% of the transcripts which were differentially expressed between the DN thymocytes from Toxf/f and Toxf/fPdcd1Cre mice, with the parameter of false discovery rate (FDR) < 0.05 and absolute fold change ≥ 2. Conclusions: Our study exhibits the detailed analysis of DN thymocyte transcriptomes with biologic replicates, generated by RNA-seq technology. The optimized data analysis here provides a framework for comparative investigations of expression profiles. The RNA-seq data allow researchers to identify the transcripts affected by TOX and to better understand the role of TOX in the development of thymocytes.
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare the difference of NGS-derived transcriptome profiling between DN thymocytes from Toxf/f and Toxf/fCd4Cre mice. Methods: mRNA profiles of DN thymocytes sorted by FACS from 3-4-week-old Toxf/f and Toxf/fCd4Cre mice were generated by deep sequencing, in triplicate, using Illumina Novaseq6000. The sequence reads that passed quality filters were analyzed at the transcript level with qRT–PCR using TaqMan and SYBR Green assays. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (Build Ensembl_release100) and identified 19,045 transcripts in the DN thymocytes of Toxf/f and Toxf/fCd4Cre mice with BWA workflow. RNA-seq data showed approximately 1.1% of the transcripts which were differentially expressed between the DN thymocytes from Toxf/f and Toxf/fCd4Cre mice, with the parameter of false discovery rate (FDR) < 0.05 and absolute fold change ≥ 2. Conclusions: Our study exhibits the detailed analysis of DN thymocyte transcriptomes with biologic replicates, generated by RNA-seq technology. The optimized data analysis here provides a framework for comparative investigations of expression profiles. The RNA-seq data allow researchers to identify the transcripts affected by TOX and to better understand the role of TOX in the development of thymocytes.
Project description:Krüppel-like factor 4 (Klf4), a transcription factor mediating context-dependent activation or repression, plays a key role in maintaining pluripotency of stem cells and in regulating cell differentiation. However, the precise function of Klf4 in T cell development and differentiation is largely unknown. Here we showed that Klf4 was highly expressed in thymocyte subsets and mature T cells, and was rapidly down-regulated in mature T cells after activation. In T cell-specific Klf4 conditional knockout mice, we observed a modest reduction of thymocytes (27%). This was due partly to the loss of Klf4 repression of Cdkn1b expression in thymocytes resulting in decreased proliferation of DN thymocytes. Together, these findings identify Klf4 as a critical regulator in T cell development. Klf4fl/fl mice (Katz et al., 2002) were crossed with CD4-Cre mice (Lee et al., 2001) to generate Klf4fl/fl-CD4-Cre+ (Klf4 conditional knockout in T cells) and Klf4fl/fl-CD4-Cre- (control) mice. Mice used for experiments were between 8 and 10 weeks old. Total RNA was extracted from freshly isolated DP+SP4 thymocytes from 3 KO mice and 3 control mice. RNA samples were labeled and hybridized to Illumina Sentrix MouseRef-8 v2 bead arrays.
Project description:We collected whole genome testis expression data from hybrid zone mice. We integrated GWAS mapping of testis expression traits and low testis weight to gain insight into the genetic basis of hybrid male sterility.