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M. A. M. Akinci, J. M. Wolosin; Identification of Genes Over-Expressed in the Limbal Stem Cell Rich Side Population (SP) via DDRT. Invest. Ophthalmol. Vis. Sci. 2007;48(13):448.
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Earlier on, using RT-PCR differential gene display (DGD) to compare the limbal (stem cell-rich) and the corneal epithelial zones , we identified genes (50+) that are over-expressed in the rabbit limbus (Li-genes; Int J. Dev Biol.,48:981 ). Independently, we have demonstrated the isolation of limbal stem cells in the form of Hoechst 33342 flow cytometry side population (SP; J Cell Sci. 118:1715). The purpose of this study was to determine whether and which of the Li-genes are in fact overexpressed in these stem cells.
Single cell suspensions of rabbit limbal epithelial cells were generated by sequential Dispase-trypsin digestion. and cultured for 24 hr. Adherent (i.e., basal) cells were stained with Hoechst 33342 and sorted by FACS into a SP (stem cell rich; ~ 1% of total) and non-SP (stem cell depleted) fractions. RNA was prepared with Tri-Reagent and used to establish relative levels of Li-gene expression in the SP and non-SP populations by real time RT-PCR. Data was normalized by the ΔΔ method using ß-actin as the invariant gene and SP/non-SP ratios (R) were calculated from duplicate experiments.Primers were designed to have highly similar potency as determined by PCR of rabbit genomic DNA, thereby allowing calculations of relative abundance (Abund.).
Out of 18 genes tested, 7 were overexpressed in the SP. All these low copy number genes had known functions that are consistent with a role in stem cell biology (see Table).
Comparison of Li vs Co gene expression by DGD led to the identification of very low copy number genes which may contribute to the stem cell phenotype.
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