Project description:CB CD34+ cells were were transduced with control (tNGFR) or CITED2 overexpression lentivectors, or control (shSCR) or shRNA CITED2 lentivectors and mRNA was isolated in order to investigate global gene expression changes upon perturbation of CITED2. CB CD34+ cells were isolated by Miltenyi miniMACS column. Cells were prestimulated in HPGM with 100 ng/ml KITL, FLT3L and TPO for 12 hrs. Cells were transduced with control (tNGFR) or CITED2 overexpression lentivectors, or control (shSCR) shRNA CITED2 lentivectors, in three rounds over 48 hrs. Transduced cells were sorted after which RNA was isolated for Illumina beadhchip arrays HT12 v4.
Project description:CB CD34+ cells were were transduced with control (tNGFR) or CITED2 overexpression lentivectors, or control (shSCR) or shRNA CITED2 lentivectors and mRNA was isolated in order to investigate global gene expression changes upon perturbation of CITED2.
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes
Project description:Gene methylation profiling of immortalized human mesenchymal stem cells comparing HPV E6/E7-transfected MSCs cells with human telomerase reverse transcriptase (hTERT)- and HPV E6/E7-transfected MSCs. hTERT may increase gene methylation in MSCs. Goal was to determine the effects of different transfected genes on global gene methylation in MSCs.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.