Project description:Mouse embryonic stem cells lines derived from different mouse strains show considerable variation concerning their culture conditions, gene targeting efficiency and germ line transmission capacity. To gain insight into the basis for this phenomenon, we generated a SAGE library of the C57BL/6 derived ES cell line Bruce-4 and compared their gene expression to 129 strain-derived cells, which in general show the best performance in gene targeting experiments. Gene Ontology (GO) analyses for functional groups of genes demonstrated considerably higher expression of clusters indicating cellular differentiation as well as cell-cell and cell-matrix adhesion in R1 than in Bruce-4 cells. At the single gene level, almost 90% of the most strongly expressed transcripts in each library were significantly differential. Regarding transcripts that have been linked to stem cell state, seven of the 20 selected genes were expressed in both cell lines, eleven showed stronger expression in Bruce-4 ES cells and only one in R1 cells. These results suggest that expression of “stemness” genes cannot be used as a sole criterion for efficiency of ES cells in gene targeting experiments. Keywords: Embryonic stem cells, SAGE, gene expression profile We generated a SAGE library of the C57BL/6-derived ES cell line Bruce-4 and compared their gene expression to an already available SAGE library of 129-derived R1 ES cells generated by Anisimov et al (GSM72878).
Project description:Mouse embryonic stem cells lines derived from different mouse strains show considerable variation concerning their culture conditions, gene targeting efficiency and germ line transmission capacity. To gain insight into the basis for this phenomenon, we generated a SAGE library of the C57BL/6 derived ES cell line Bruce-4 and compared their gene expression to 129 strain-derived cells, which in general show the best performance in gene targeting experiments. Gene Ontology (GO) analyses for functional groups of genes demonstrated considerably higher expression of clusters indicating cellular differentiation as well as cell-cell and cell-matrix adhesion in R1 than in Bruce-4 cells. At the single gene level, almost 90% of the most strongly expressed transcripts in each library were significantly differential. Regarding transcripts that have been linked to stem cell state, seven of the 20 selected genes were expressed in both cell lines, eleven showed stronger expression in Bruce-4 ES cells and only one in R1 cells. These results suggest that expression of “stemness” genes cannot be used as a sole criterion for efficiency of ES cells in gene targeting experiments. Keywords: Embryonic stem cells, SAGE, gene expression profile
Project description:PURPOSE: To provide a detailed gene expression profile of the normal postnatal mouse cornea. METHODS: Serial analysis of gene expression (SAGE) was performed on postnatal day (PN)9 and adult mouse (6 week) total corneas. The expression of selected genes was analyzed by in situ hybridization. RESULTS: A total of 64,272 PN9 and 62,206 adult tags were sequenced. Mouse corneal transcriptomes are composed of at least 19,544 and 18,509 unique mRNAs, respectively. One third of the unique tags were expressed at both stages, whereas a third was identified exclusively in PN9 or adult corneas. Three hundred thirty-four PN9 and 339 adult tags were enriched more than fivefold over other published nonocular libraries. Abundant transcripts were associated with metabolic functions, redox activities, and barrier integrity. Three members of the Ly-6/uPAR family whose functions are unknown in the cornea constitute more than 1% of the total mRNA. Aquaporin 5, epithelial membrane protein and glutathione-S-transferase (GST) omega-1, and GST alpha-4 mRNAs were preferentially expressed in distinct corneal epithelial layers, providing new markers for stratification. More than 200 tags were differentially expressed, of which 25 mediate transcription. CONCLUSIONS: In addition to providing a detailed profile of expressed genes in the PN9 and mature mouse cornea, the present SAGE data demonstrate dynamic changes in gene expression after eye opening and provide new probes for exploring corneal epithelial cell stratification, development, and function and for exploring the intricate relationship between programmed and environmentally induced gene expression in the cornea. Keywords: other
Project description:A transcriptome study in mouse hematopoietic stem cells was performed using a sensitive SAGE method, in an attempt to detect medium and low abundant transcripts expressed in these cells. Among a total of 31,380 unique transcript, 17,326 (55%) known genes were detected, 14,054 (45%) low-copy transcripts that have no matches to currently known genes. 3,899 (23%) were alternatively spliced transcripts of the known genes and 3,754 (22%) represent anti-sense transcripts from known genes. Mouse hematopoietic stem cells were purified from bone marrow cells using negative and positive selection with a Magnetic-Activated Cell Sorter (MACS). total RNA and mRNA were purified from the purified cells using Trizol reagent and magnetic oligo dT beads. Double strand cDNAs were synthesized using a cDNA synthesis kit and anchored oligo dT primers. After NlaIII digestion, 3’ cDNAs were isolated and amplified through 16-cycle PCR. SAGE tags were released from the 3’ cDNA after linker ligation. Ditags were formed, concatemerized and cloned into a pZERO vector. Sequencing reactions were performed with the ET sequencing terminator kit. Sequences were collected using a Megabase 1000 sequencer. SAGE tag sequences were extracted using SAGE 2000 software.
Project description:A transcriptome study in mouse hematopoietic stem cells was performed using a sensitive SAGE method, in an attempt to detect medium and low abundant transcripts expressed in these cells. Among a total of 31,380 unique transcript, 17,326 (55%) known genes were detected, 14,054 (45%) low-copy transcripts that have no matches to currently known genes. 3,899 (23%) were alternatively spliced transcripts of the known genes and 3,754 (22%) represent anti-sense transcripts from known genes.