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Shweta Singhal, Lauren M. James, Bhairavi Bhatia, Hari Jayaram, Peng T. Khaw, G Astrid Limb; Construction and Characterization of a Human BRN3B Transcriptional Reporter to Identify Committed RGC Precursors In Vitro and In Vivo. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2247.
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BRN3B is expressed in post mitotic retinal ganglion cell precursors and is a marker of retinal ganglion cell fate commitment in differentiating retinal progenitors. This Pou4 domain transcription factor was used to create a BRN3B based transcriptional reporter to identify committed RGC precursors amongst human Müller stem cells induced to undergo neural retinal differentiation in vitro.
A 1.6kb putative human BRN3B promoter sequence was introduced into a promoterless GFP vector (pEGFP-1) upstream of the GFP coding region such that GFP would be expressed under control of the BRN3B promoter region.Following sequence verification, the reporter was stably transfected into adult Müller stem cells of human origin and its expression pattern studied.
Stably transfected and green (BRN3B reporter positive) cells separated from non green (BRN3B reporter negative) cells by FACS sorting showed significantly higher levels of BRN3B and ATOH7 mRNA expression. They co-stained with the BRN3B antibody and in some cases demonstrated striking neuronal morphology. They were also found to be largely post mitotic (less than 10% Ki67 positive) and expressed higher levels (nearly 70% positive) of ISL1 (another RGC marker) compared to non-green cells. No GFP expression was seen when the reporter was transfected into human trabecular meshwork fibroblasts, which do not express BRN3B. Upon transplantation intravitreally in rat retinae, the transfected cells also showed GFP positivity.
A human BRN3B transcriptional reporter can identify committed retinal ganglion cell precursors and would make a useful tool to distinguish committed RGC precursors amongst Müller stem cells induced to differentiate into retinal neurons in vitro, as well as to track the behaviour of committed RGC precursors after transplantation in vivo.
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