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Microarray analysis on E8.25 definitive and visceral endoderm

Embryos (E8.25) were dissected into embryonic and extraembryonic tissues from which single cells were isolated, antibody-labeled, and purified through FACS - by germ layer:

  • Embryonic endoderm
  • Embryonic ectoderm
  • Embryonic mesoderm
  • Extraembryonic endoderm
  • Extraembryonic mesoderm

Isolated RNA was used to synthesize cDNA, which was hybridized (in triplicate) to Affymetrix microarray chips (Mouse Genome 430 2.0 GeneChip). Resulting data was analyzed by GenePattern (Broad Institute) and the analysis data is provided here. Raw data will become available at a later date.

Data

Excel spreadsheet Microarray analysis spreadsheet (GenePattern)

Note: Annotations, methods and additional information is available on the second worksheet. The data is on the first worksheet.

Added on April 23, 2007

Contributors and Contact Information

Sherwood R.I. (Contact: rsherw@fas.harvard.edu ), lead author, PhD Candidate, Melton Lab, Department of Molecular and Cell Biology, Harvard University
Jitianu C. former Graduate student, Golub lab, Broad Institute of Harvard and MIT
Cleaver O. former Postdoc, Melton Lab, Department of Molecular and Cell Biology, Harvard University
Shaywitz D.A. former Postdoc, Melton Lab, Department of Molecular and Cell Biology, Harvard University
Lamenzo J.O. Project Manager, Melton Lab, Department of Molecular and Cell Biology, Harvard University
Chen A.E. Postdoc, Melton Lab, Department of Molecular and Cell Biology, Harvard University
Golub T.R. PI, Golub lab, Broad Institute of Harvard and MIT
Melton D.A. PI, Melton Lab, Department of Molecular and Cell Biology, Harvard University

Methods

  1. E8.25 (2-6 somite) embryos were dissected into embryonic and extraembryonic tissues based upon anatomy, and dissociated into single cells with trypsin,
  2. Cells were then labeled with EpCAM and DBA surface antibodies and purified through FACS by germ layer,
  3. Total RNA was extracted, cDNAs synthesized and hybridized in triplicate to Affy chips (Mouse Genome 430 2.0 GeneChip),
  4. Resulting data were analyzed using Gene Pattern (Broad Institute),
  5. Values are the averages of three replicates in arbitrary units.

Reference

Prospective isolation and global gene expression analysis of definitive and visceral endoderm

Sherwood RI, Jitianu C, Cleaver O, Shaywitz DA, Lamenzo JO, Chen AE, Golub TR, Melton DA.
Department of Molecular and Cellular Biology, Harvard Stem Cell Institute, Harvard University, 7 Divinity Ave, Cambridge, MA 02138, USA.

In spite of the therapeutic importance of endoderm derivatives such as the pancreas, liver, lung, and intestine, there are few molecular markers specific for early endoderm. In order to identify endoderm-specific genes as well as to define transcriptional differences between definitive and visceral endoderm, we performed microarray analysis on E8.25 definitive and visceral endoderm. We have developed an early endoderm gene expression signature, and clarified the transcriptional similarities and differences between definitive and visceral endoderm. Additionally, we have developed methods for flow cytometric isolation of definitive and visceral endoderm. These results shed light on the mechanism of endoderm formation and should facilitate investigation of endoderm formation from embryonic stem cells.

This content was last modified on April 23, 2007.