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Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human AFP protein, residues surrounding Gly138 of human FoxA2/HNF3β protein, residues near the amino terminus of human GATA-6 protein, the sequence of human HNF4α protein, a recombinant protein corresponding to the PDGF receptor α extracellular domain, or residues surrounding Ala311 of human Sall4 protein. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human N-cadherin protein or near the amino terminus of human EOMES protein. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.
Two endodermal lineages develop during mammalian embryogenesis, the primitive endoderm of the blastocyst stage embryo and the definitive endoderm at gastrulation. The primitive endoderm gives rise to extra-embryonic lineages encompassing the visceral and the parietal endoderm. The definitive endoderm contributes to the respiratory and gastrointestinal tracts by forming the epithelial lining of the trachea, esophagus, lungs, stomach and intestines, and is a major component of many glands, including thyroid, thymus, pancreas and liver (1). Understanding molecular mechanisms that regulate early endodermal fates is seminal for the advance of stem cell research as they connect the transition from pluripotency to endoderm specification during mammalian development and contribute to the generation of clinically relevant cell types. FoxA2/HNF3β is a transcription factor essential for development of the endoderm and midline structures in mouse embryos (2,3). EOMES acts during gastrulation to promote the specification of the definitive endoderm (4). Markers of hepatic differentiation in the endoderm include expression of α-fetoprotein (AFP) and N-cadherin (5,6). HNF4α is involved in the differentiation of the visceral endoderm. GATA-6 lies upstream of HNF4 in a transcriptional cascade that regulates differentiation of the visceral endoderm and is also required for the establishment of the endodermally derived bronchial epithelium (7). Sall4 is required for the formation of the primitive endoderm from inner cell mass. It has been reported that extra-embryonic stem cell lines cannot be formed in Sall4-deficient blastocysts (8). PDGF receptor α is expressed in primitive endoderm derivatives throughout embryogenesis (9).
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