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M.A. Shatos, H. Klassen, P.H. Schwartz, J. Doherty, B. Ziaeian, I. Kirov, H. Nethercott, M. Samuel, R.S. Prather, M.J. Young; Isolation of progenitor cells from retina and brain of the GFP–transgenic pig . Invest. Ophthalmol. Vis. Sci. 2004;45(13):5406.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To isolate progenitor cells from the developing retina (RPCs) and brain (BPCs) of green fluorescent protein (GFP)–transgenic pigs and examine gene expression of these cells. Methods: A timed–pregnant wild–type sow was generated from a heterozygous GFP–transgenic male. At embryonic day 60 (E60, gestation 114 days), fetuses were removed and retinas and brains harvested from GFP+ donors. Retinal and brain tissues were enzymatically digested and cultured in several combinations of growth media (EGF, PDGF, bFGF, with and without laminin or fibronectin). The resulting cells were characterized by immunocytochemistry (ICC), western blot and RT–PCR. Results: At E60, many of the brain cells, but few of the retinal cells, expressed GFP, although other ocular structures were GFP+. With time in culture, RPCs gradually became GFP+. After passaging with trypsin and propagation, nearly all BPCs and RPCs expressed the GFP transgene. ICC of RPCs and BPCs revealed expression of Ki–67, nestin, doublecortin, GFAP, NCAM, Sox2, and b–111 tubulin. RT–PCR showed expression of Dach1, Hes1, Pax6, Six3 and Six6. Upon differentiation in 10% serum, both cell cultures expressed GFAP, NF200, NFM and MAP5, while only RPCs expressed rhodopsin and recoverin (by ICC, western blot and RT–PCR), suggesting rod photoreceptor differentiation. Conclusions: RPCs and BPCs can be readily isolated from the embryonic pig CNS and, upon expansion in vitro, widespread expression of the GFP transgene can be seen. As RPCs can be induced to differentiate into photoreceptor cells, grafting these GFP+ cells into the porcine retina would be of considerable interest.
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