Sylvie Tondeur | PubMed Health

PubMed2007

A meta-analysis of human embryonic stem cells transcriptome integrated into a web-based expression atlas.

Microarray technology provides a unique opportunity to examine gene expression patterns in human embryonic stem cells (hESCs). We performed a meta-analysis of 38 original studies reporting on the transcriptome of hESCs. We determined that 1,076 genes were found to be overexpressed in hESCs by at least three studies when compared to differentiated cell types, thus composing a "consensus hESC gene list." Only one gene was reported by all studies: the homeodomain transcription factor POU5F1/OCT3/4. The list comprised other genes critical for pluripotency such as the transcription factors NANOG and SOX2, and the growth factors TDGF1/CRIPTO and Galanin. We show that CD24 and SEMA6A, two cell surface protein-coding genes from the top of the consensus hESC gene list, display a strong and specific membrane protein expression on hESCs. Moreover, CD24 labeling permits the purification by flow cytometry of hESCs cocultured on human fibroblasts. The consensus hESC gene list also included the FZD7 WNT receptor, the G protein-coupled receptor GPR19, and the HELLS helicase, which could play an important role in hESCs biology. Conversely, we identified 783 genes downregulated in hESCs and reported in at least three studies. This "consensus differentiation gene list" included the IL6ST/GP130 LIF receptor. We created an online hESC expression atlas, http://amazonia.montp.inserm.fr, to provide an easy access to this public transcriptome dataset. Expression histograms comparing hESCs to a broad collection of fetal and adult tissues can be retrieved with this web tool for more than 15,000 genes.

Cell Differentiation↗

PubMed2004

Overcoming bacterial DNA contamination in real-time PCR and RT-PCR reactions for LacZ detection in cell therapy monitoring.

Since the introduction of the polymerase chain reaction the presence of contaminating bacterial DNA in the Taq polymerase preparations has hampered the use of this technique in microbiology. Lately, this inconvenience has equally impeded gene quantification in the field of cell or gene therapy, where bacterial genes such as LacZ are often used as tags to detect vectors or cells after their injection in the recipient organism. Several means to overcome the DNA contamination of Taq Polymerase have been reported with variable degrees of decontamination efficiency and alteration of the PCR reaction. Here we propose two protocols to efficiently quantify DNA or RNA from the LacZ gene by real-time PCR using either decontamination by low concentrations of DNAse I prior to PCR amplification or a highly purified Taq Polymerase which is devoid of detectable contamination.

Animals↗