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Next, 4.0 μg of total RNA for each cell type was DNase-treated (DNA-free, Ambion, Austin, TX) and divided in half for first-strand cDNA synthesis with or without reverse transcriptase by using the SuperScript II kit (Invitrogen). Total RNA was isolated from P/Sox2-EGFP-expressing ESCs and NSCs as described for expression profiling. P/Sox2-GIP ESCs were used as a positive control for ICM adherence and chimeric contribution. To detect nontelencephalic chimerism, embryos were fixed for 10 min at room temperature in 0.2% glutaraldehyde, washed three times in detergent rinse, and then stained in 5-bromo-4-chloro-3-indolyl β- d-galactoside (X-Gal) reaction solution overnight at 4☌ ( 20). Embryos were collected at E9.5 and analyzed for EGFP expression by using an inverted fluorescence microscope. Blastocysts were transferred into the uterine horns of pseudopregnant females. Blastocysts were allowed to reexpand for 2-4 h at 37☌, at which time they were examined by using an inverted fluorescence microscope to assess the number of blastocysts that had cells attached to the inner cell mass (ICM). Cells were found primarily as single cells or doublets the next day, and ≈12 were injected into each blastocyst. P/Sox2-EGFP-positive cells were isolated from E14 telencephalon or cultured neural stem/progenitor cells by using FACS and were grown overnight in B27 media with growth factors. Materials and MethodsĬhimera Production Assay.
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Third, a Sox2 regulatory element has been described that expresses in ESCs as well as in neural progenitor populations ( 17). This property is useful for further characterizing the phenomenon of unexpected plasticity in NSC populations ( 7). Second, outside of these pluripotent cells, Sox2 is a neural restricted gene ( 16) and therefore unequivocally identifies a neural tissue of origin. Furthermore, Sox2 is known to act cooperatively at several promoters with Oct4 ( 10- 13), and Oct4 is believed to be the master regulator of the pluripotent state ( 14, 15). First, Sox2 is strongly associated with the pluripotent phenotype, because it is expressed in ESCs and gene-deleted mice exhibit embryonic lethality at implantation ( 8, 9). The transcription factor Sox2 has several ideal properties for use in comparing ESCs and NSCs. Because methods for isolating SCs are varied and not well developed, we sought an approach that would allow the isolation of both embryonic SCs (ESCs) and neural SCs (NSCs) to investigate the genetic and functional relationships between these SC classes.
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