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. 2011 Oct 13;21(9):1571–1586. doi: 10.1089/scd.2011.0370

FIG. 3.

FIG. 3.

Characterization of rat ESCs derived from transgenic rats. (A–C) Detection of transgenes genes in ESCs derived from transgenic F344 rats. (A) X-gal histochemistry (X-gal Histochem) to detect β-gal in ESCs (line 2, passage 11) derived from β-gal transgenic rat. (B) Immunocytochemical staining for β-gal protein (β-gal Immuno, red) in rat ESC line 2 counterstained with DAPI for DNA (blue). Scale bar is 100 μm. (C) PCR on genomic DNA extracted from rat ESC lines 61, 62, 1, 2, and 3, to detect human placental AP (left) or β-gal transgene expression (β-gal, middle and right). ESC lines 61 and 61 were derived from the human placental AP transgenic rats and lines 1, 2, and 3 were derived from the β-gal transgenic rats. Lane W, water (negative control). (D) Embryoid bodies (EBs) derived from ESCs. Left: EBs formed using the hanging-drop method for 8 days. Right: EBs formed using suspension culture method for 8 days. EBs were derived from line 2 (β-gal F344 ESCs) at passage 12. Scale bar 100 μm. (E) RT-PCR characterization of pluripotency transcription factor expression in EBs. EBs cultured in the presence of bone morphogenetic protein 4 (BMP4) for 5 or 10 days show progressive down-regulation of Oct4 (lane1), and Nanog (lane 2) and not the control gene (PBGD, lane 3). Top: ESCs maintained in 2i medium to prevent differentiation (undifferentiated control). Middle and bottom: EBs maintained in medium with BMP4 for 5 days (middle) or 10 days (bottom). (F) ESCs derived from transgenic F344 rats form teratoma after injection into parental strain F344 rats. ESCs from Line 1 were injected subcutaneously into parental strain F344 rats. Ten weeks later, a tumor was palpated at the injection site (tumor in situ, left). The tumor was isolated, collected, and histologically processed by the KSU veterinary medicine diagnostic laboratory. Hematoxylin and eosin staining (right) of paraffin sections showed hair follicles, sweat glands, adipose tissue, skeletal muscle and hyaline cartilage (right at top), and respiratory epithelium (right, bottom). Thus, ESC-derived tumors formed tissues from all 3 germ layers. Color images available online at www.liebertonline.com/scd