Project description:Obesogens such as tributyltin (TBT) are xenobiotic compounds that promote obesity, in part by distorting the normal balance of lipid metabolism. The obesogenic effects of TBT can be observed in directly exposed (F1 and F2 generations) and also subsequent generations (F3 and beyond) that were never exposed. To address the effects of TBT exposure on germ cells, we exposed pregnant transgenic OG2 mouse dams (F0), which specifically express EGFP in germline cells, to an environmentally relevant dose of TBT throughout gestation through drinking water. When fed with a high fat diet (HFD), F3 male offspring of TBT-exposed F0 dams (TBT-F3) accumulated much more body fat than did Control-F3 males. TBT-F3 males also lost more body fluid and lean compositions than did Control-F3 males. Expression of genes involved in transcriptional regulation or mesenchymal differ-entiation was upregulated in somatic cells of TBT-F1 (but not TBT-F3) E18.5 fetal testes, and promoter-associated CpG islands were hyper-methylated in TBT-F1 somatic cells. Global mRNA expression of protein-coding genes in F1 or F3 fetal testicular cells was unaffected by F0 exposure to TBT; however, expression of a subset of endogenous retroviruses was significantly affected in F1 and F3. We infer that TBT may directly target testicular somatic cells in F1 testes to irreversibly affect epigenetic suppression of endogenous retroviruses in both germline and somatic cells.
Project description:Transcriptional profiling of transformed Ba/F3 cells by myeloproliferative neoplasm-associated JAK2 V617F mutant comparing control Ba/F3 cells expressing wild type JAK2. Two-condition experiment, WT cells vs. VF cells. One replicate per array.
Project description:In order to identify patterns of gene expression associated with biological effects in THP-1 cells induced by F3, we performed a transcriptomic analysis on the THP-1 control and F3-treated THP-1 cells by oligonucleotide microarray
Project description:SPAG9-JAK2 is a novel fusion gene identified in a pediatric patient with Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL). In this study, we performed functional analysis of the SPAG9-JAK2 fusion to establish molecular targeted therapy. Ba/F3 cells expressing SPAG9-JAK2 generated by retroviral transduction (Ba/F3-SPAG9-JAK2), proliferated in the absence of IL-3, and exhibited constitutive phosphorylation of the tyrosine residues in the JAK2 kinase domain of the fusion protein and STAT3/STAT5. Mutation of tyrosine residues in the JAK2 kinase domain (SPAG9-JAK2 mut) abolished IL-3 independence, but had no influence on STAT3/STAT5 phosphorylation levels. Gene expression analysis revealed that Stat1 was significantly up-regulated in Ba/F3-SPAG9-JAK2 cells. STAT1 was also phosphorylated in Ba/F3-SPAG9-JAK2 but not SPAG9-JAK2 mut cells, suggesting that STAT1 is key for SPAG9-JAK2-mediated cell proliferation. Consistently, STAT1 induced expression of the anti-apoptotic proteins, BCL-2 and MCL-1, as did SPAG9-JAK2, but not SPAG9-JAK2 mut. Ruxolitinib abrogated Ba/F3-SPAG9-JAK2-mediated proliferation in vitro, but was insufficient in vivo. Venetoclax (a BCL-2 inhibitor) or AZD5991 (an MCL-1 inhibitor) enhanced the effects of ruxolitinib on Ba/F3-SPAG9-JAK2 in vitro. These findings suggest that activation of the JAK2-STAT1-BCL-2/MCL-1 axis contributes to SPAG9-JAK2-related aberrant growth promotion. BCL-2 or MCL-1 inhibition is a potential therapeutic option for B-ALL with SPAG9-JAK2 fusion.
Project description:Ba/F3 cells were transformed after transfection with CRISPR/CAS9 + gRNA vs target gene. Oligoclonal cell population was flow sorted into single cells and processes for RNAseq.
Project description:Embryonic exposure to the endocrine disruptor vinclozolin during gonadal sex determination appears to promote an epigenetic reprogramming of the male germ line that is associated with transgenerational adult-onset disease states. Transgenerational effects on the embryonic day 16 (E16) testis demonstrated reproducible changes in the testis transcriptome for multiple generations (F1-F3). The expression of 196 genes was found to be influenced, with the majority of gene expression being decreased or silenced. Dramatic changes in the gene expression of methyltransferases during gonadal sex determination were observed in the F1 and F2 vinclozolin generation (E16) embryonic testis, but the majority returned to control-generation levels by the F3 generation. The most dramatic effects were on the germ-line-associated Dnmt3A and Dnmt3L isoforms. Observations demonstrate that an embryonic exposure to vinclozolin appears to promote an epigenetic reprogramming of the male germ line that correlates with transgenerational alterations in the testis transcriptome in subsequent generations. Experiment Overall Design: E16 Testis RNA samples from F1, F2, F3 generation control groups are compared to F1, F2, F3 generation vinclozolin treated groups
