Open Access Research

A mouse embryonic stem cell bank for inducible overexpression of human chromosome 21 genes

Rossella De Cegli1, Antonio Romito12, Simona Iacobacci1, Lei Mao3, Mario Lauria1, Anthony O Fedele14, Joachim Klose3, Christelle Borel5, Patrick Descombes6, Stylianos E Antonarakis5, Diego di Bernardo1, Sandro Banfi1, Andrea Ballabio1 and Gilda Cobellis17*

Author Affiliations

1 Telethon Institute of Genetics and Medicine, Via P. Castellino 111, Napoli, 80131, Italy

2 Current address: Université Paris Diderot - Paris 7, Paris Cedex 13, Paris, 75205, France

3 Institut für Humangenetik Charité, Campus Virchow-Klinikum, Universitätsmedizin Berlin, Augustenburger Platz 1, Berlin, D-13353, Germany

4 Current address: Lysosomal Diseases Research Unit, SA Pathology, 72 King William Road, North Adelaide, South Australia, 5006, Australia

5 Department of Genetic Medicine and Development, University of Geneva Medical School, 1 rue Michel-Servet, Geneva, CH-1211, Switzerland

6 Genomics Platform, University of Geneva Medical School, 1 rue Michel-Servet, Geneva, CH-1211, Switzerland

7 Current address: Dipartimento di Patologia Generale, Seconda Universita' di Napoli, Via De Crecchio 7, Napoli, 80100, Italy

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Genome Biology 2010, 11:R64  doi:10.1186/gb-2010-11-6-r64

Published: 22 June 2010

Additional files

Additional file 1:

Identification and validation of inducible/exchangeable recombinant mES clones. (a) Recombinant mES clones were identified by PCR analysis. (b) q-PCR analysis and Luciferase assays using Dual Luciferase Reporter Assay System was performed on mES clones overexpressing the firefly luciferase (Luc) gene. The system was activated upon the removal of Tc (after 17, 24, 39 and 48 hours) from the medium. Protein extracts of mES cells were prepared at the same time points and luminescence quantified. (c) q-PCR analysis and YFP fluorescence assay to detect the expression of the YFP reporter. (d) Expression of mES cells overexpressing Luc after 24 hours from the complete removal of Tc from the medium; the degree of induction was easily manipulated by titrating the Tc. (e) Expression profile (q-PCR) of the pluripotency gene Oct3/4, and of markers of the mesoderm (Brachyury), ectoderm (Gfap) and endoderm (Afp) during differentiation of EB3 and of the parent cell line (E14).

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Additional file 2:

List of 32 genes overexpressed in mouse ES cells. In this table we list the 32 genes selected to be integrated in the Rosa26 locus and overexpressed using the Tet-off system in mES cells.

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Additional file 3:

Time course of induction of three clones (biological replicates) selected for each gene. In this table we report the time course of the induction of mES clones that overexpress the 32 ORFs. For each gene, three drug-resistant mES biological replicates, whose names are indicated in the specific column, were selected to be tested for their sensitivity to Tc removal from the medium.

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Additional file 4:

Primer pairs used in q-PCR.

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Additional file 5:

Comparison of relative expression levels for 20 genes in the EB3 parental cell line and in the inducible clones at 0 hours of induction by multiple statistical t-tests. In this table we show the comparison of the relative expression of 20 genes (the 13 transcription factors, the single transcriptional activator and the 6 kinases) in the EB3 cell line versus the corresponding transgenic inducible clones (in the biological replicates) grown in the presence of Tc (0 hours of induction).

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Additional file 6:

Complete list of differentially expressed genes following the overexpression of Aire, one of the effective genes.

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Additional file 7:

Complete list of differentially expressed genes following the overexpression of Erg, one of the effective genes.

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Additional file 8:

Complete list of differentially expressed genes following the overexpression of Nrip1, one of the effective genes.

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Additional file 9:

Complete list of differentially expressed genes following the overexpression of Olig2, one of the effective genes.

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Additional file 10:

Complete list of differentially expressed genes following the overexpression of Pdxk, one of the effective genes.

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Additional file 11:

Complete list of differentially expressed genes following the overexpression of Runx1, one of the effective genes.

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Additional file 12:

Complete list of differentially expressed genes following the overexpression of Sim2, one of the effective genes.

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Additional file 13:

Summary of q-PCR validation of microarray data. In this table we show the validation by q-PCR of the differential expression of a subset of the most up-regulated and down-regulated genes detected by microarray analysis of the seven effective genes, as ranked by differential expression ratio.

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Additional file 14:

A complete list of significantly enriched GO terms for the seven effective genes.

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Additional file 15:

List of all the mouse orthologs of HSA21 genes sorted according to their basal expression level in mES cells (from the most to the least expressed).

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Additional file 16:

Summary of results derived from the comparison between the analysis of mES overexpressing effective clones and the transchromosomic Tc1 mouse line.

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Additional file 17:

Comparison of overexpression experiments with the transcriptional response of the transchromosomic tc1 mouse line. X-Y graphs comparing the transcriptional response of Tc1 with the response obtained in the individual overexpression experiments. Each dot represents a gene whose expression was statistically significant in both the Tc1 and the indicated overexpression experiment. The x axis corresponds to the log of the Tc1 ratio (trisomic versus wild type), and the y axis corresponds to the log of the ratio in the overexpression experiment (induced versus non-induced clone). The ratio of same-sign over total dots is reported for each graph.

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Additional file 18:

A complete list of GO terms significantly enriched in the subsets of genes differentially expressed after overexpression of 11 out of 13 silent genes.

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Additional file 19:

GO enrichment analysis for five (Bach1, Dscr1-Rcan1, DYRK1A, Gabpa and SNF1LK) out of thirteen silent genes, as assessed by microarray analysis. In this table we report the GO enrichment analysis for five out of thirteen silent genes (Bach1, Dscr1-Rcan1, DYRK1A, Gabpa, SNF1LK); supporting references for a subset of significant biological processes identified by the GO analysis are given.

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Additional file 20:

Differential protein expression variation in mES cells overexpressing Runx1. In this table we report the complete list of the proteins whose expression changed following the induction of Runx1

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Additional file 21:

Primer pairs used in PCR.

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Additional file 22:

Raw data of the proteomic analysis, using 2DGE, following the induction of two Runx1 overexpressing clones (E6 and E7). Three technical repeats were performed for each biological replicate: this comprises 6 gels for E6 and 6 gels for E7. For each clone we ran three replicates for t0 (control) and three replicates for t48 hours. Corresponding t0 and t48 hour samples were always run simultaneously in the same chamber to ensure gel pattern comparability. The detailed spot quantification data are provided in this table. We list the relative volume of each spot on each individual 2DGE gel, together with other spot parameters, such as pixel spot volume, x and y coordinates of spots on the fusion gel image, as well as spot quality index.

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