Project description:E-cadherin upregulation is an early event of reprogramming of fibroblasts to induce pluripotent stem cells (iPS). Knocking down of E-cadherin by shRNA impairs iPS generation, though some colonies with great morphorlogical difference to shRNA control colonies remain. To illustrate the molecular and functional difference between shECAD iPS clones and shRNA control iPS clones, three respective iPS clones (shECAD 4,8,9 and Ctrl 2,3,4) were derived and DNA microarrays were run to analyze the transcriptional profile of these clones.
Project description:We used heterokaryon cell fusion based reprogramming and identified the cytokine IL6 as a potential regulator of reprogramming to pluripotency. We generated iPS clones using the four reprogramming factors (4F) Oct4, Klf4, Sox2, and c-Myc. In addition, iPS clones were generated using only three factors (3F: Oct4, Klf4, amd Sox2) with the addition of the cytokine IL6 to reprogramming culture conditions. Global RNA-Seq of the 3F + IL6 derived iPS clones was done for comparison with 4F-derived iPS clones, mouse embryonic stem cells and mouse embryonic fibroblasts.
Project description:LRP1B remains one of the most altered genes in cancer, although its relevance in cancer biology is still unclear. Recent advances in gene editing techniques, particularly CRISPR/Cas9 systems, offer new opportunities to evaluate the function of large genes, like LRP1B. Using a dual sgRNA CRISPR/Cas9 gene editing approach, this study aimed to assess the impact of disrupting LRP1B in glioblastoma cell biology. Four sgRNAs were designed for the dual targeting of two LRP1B exons (1 and 85). The U87 glioblastoma (GB) cell line was transfected with CRISPR/cas9 PX459 vectors. To assess LRP1B gene alterations and expression, PCR, Sanger DNA sequencing, qRT-PCR were carried out. Three clones (clones B9, E6 and H7) were further evaluated. All clones presented altered cellular morphology, increased cellular and nuclear size and changes in ploidy. Two clones (E6 and H7) showed a significant decrease in cell growth, both in vitro and in the in vivo CAM assay. Proteomic analysis of the clones’ secretome identified differentially ex-pressed proteins, that had not been previously associated with LRP1B alterations. This study demonstrates that the dual sgRNA CRISPR/Cas9 strategy can effectively edit LRP1B in GB cells, providing new insights into the impact of LRP1B deletions in GBM biology.
Project description:Microarray hybridization was used to compare RNA from mouse brains with opposite genotypes at the Mvb1 (Nxf1) modifier locus for known alternative processing events. 6 samples of total brain RNA, from 3 littermate pairs, were hybridized to splicing-sensitive microarrays *Addendum Depending on the analysis software used, these CEL files may not load correctly using default parameters. This is due to the custom chip type of MJAY not being used during the array scanning step. There are three workarounds known for this problem so far. 1) If using APT, use multiple --chip-type parameters. Specifically, --chip-type mjay --chip-type MJAY --chip-type MoEx-1_0-st-v1.1sq 2) Edit the CEL file by converting to text using the APT command apt-cel-convert, then replacing the MoEx-1_0-st-v1.1sq in the DatHeader line with MJAY (all caps). 3) Edit the .pgf, .clf, and antigenomics.bmp files to use the MoEx-1_0-st-v1.1sq array instead of MJAY for the chip_type and lib_set_name options. (works on AltAnalyze software)
Project description:Comparison of gene expression of KR Primary and KR 1 cell lines KR Primary cells are established from lung tumors of Kras mice. This cell line was injected into B6 mice intratracheally and then KR 1 cell line was established.
Project description:Purpose: A method for mapping chromatin accessibility genome-wide, to reveal chromatin accessibility in Intestinal stem cells. Methods: Intestinal stem cells(Lgr5-high cells) were sorted by flow cytometry from wild type mice. The samples were prepared in duplicate. HISAT2 was used to align the sequences to the mouse genome and generate bam files. bamCoverage was used to generate bigwig files from bam files. MACS2 (v2.2.5) was used for peak calling and to generate bed files from aligned reads. Conclusions: ATAC-seq analysis confirmed that Fosb binding sites in Chip-seq assay were correlated with the chromatin accessibility .
