Project description:HES2 ESC were infected with a lentivirus construct containing either CD30 (TNFRSF8) extracellular deficient variant or GFP, using the ViraPower™ Promoterless Lentiviral Gateway® Kits (Invitrogen). HES2 cells were recovered and grown for 19 passages. HES2 were commonly grown on Mouse Embryonic Feeder cells in the presence of KSOR media. Before collection of RNA, ES cells were FACs sorted with antibodies to GCMT-2 and CD9 to separate ES cells from differentiated cells and mouse feeders. The experiment was repeated in triplicate. The final aim was to deduce the effect of overexpressing the variant form of CD30 on gene expression in HES2 cells.
Project description:HES2 ESC were infected with a lentivirus construct containing either CD30 (TNFRSF8) extracellular deficient variant or GFP, using the ViraPower� Promoterless Lentiviral Gateway® Kits (Invitrogen). HES2 cells were recovered and grown for 19 passages. HES2 were commonly grown on Mouse Embryonic Feeder cells in the presence of KSOR media. Before collection of RNA, ES cells were FACs sorted with antibodies to GCMT-2 and CD9 to separate ES cells from differentiated cells and mouse feeders. The experiment was repeated in triplicate. The final aim was to deduce the effect of overexpressing the variant form of CD30 on gene expression in HES2 cells. Three consecutive passages of HES2 cells over-expressing either CD30V or GFP were grown and subject to FACs sorting, collection and microarray. A total of 6 samples, (3 replicates of these 2 populations) were used in the experiment.
Project description:Purpose: NGS can be used for system-based analysis of cellular pathways. The goal of this study was to identify genes and pathways which are activated in AB8 podocytes due to Crb2 S267A overexpression. Methods: AB8 cells were modified using lentiviral transduction with pIND21_hCrb2+GFP_S267A. After selection with puromycin, cells were induced with doxycycline for 48h to overexpress Crb2 S267A variant. Non-induced cells were used as controls. RNA-seq data from AB8 cells overexpressing Crb2 S267A variant for 48h (+dox) were compared with non-induced control cells (-dox) (n=4). Activated pathways (GO terms) were identified with DAVID bioinformatics tool. Results: RNAseq data revealed activation of ER stress response, chaperon folding genes and pathways. Misfolded Crb2 variant induces cellular ER stress compared to control cells.
Project description:Methylated DNA binding protein 2 (MBD2) has been shown to bind specific methylated promoters and suppress transcription. Here we systematically investigate MBD2 suppression by overexpressing MBD2 in MCF-10A cells and generating gene expression profiles of overexpressing cells and normal MCF-10A cells.