Next, hESC lines stably expressing each reporter construct were generated to follow promoter activity during RPE differentiation. Reporter hESC lines were differentiated to RPE by using the 14-day differentiation protocol.
9 Because expression of endogenous
RAX,
OTX2, and
MITF genes has been characterized,
9 this system allowed us to further test reporter specificity. No reporter fluorescence was observed in any stable hESC lines at day 0 (
Fig. 2A). On day 4 to 6, RAX-mCherry and OTX2-mCherry fluorescence became visible, then faded out by day 10 to 14 (
Fig. 2A), and endogenous levels of
RAX and
OTX2 gene expression showed a similar trend (
Fig. 2B). On day 6 to 14, MITF-D–mCherry fluorescence became observable (
Fig. 2A) at the same time that levels of endogenous
MITF were also detectable (
Fig. 2B), consistent with a role for MITF-D during optic vesicle formation and in mature RPE.
9,36 RPE65 is a marker of mature RPE,
4 and neither RPE65-mCherry reporter fluorescence nor endogenous
RPE65 gene expression was detectable from day 0 to 14 (
Figs. 2A,
2B), as cells at this stage are thought to be RPE progenitors/immature RPE
9 (for RPE65 reporter expression in mature RPE, see
Fig. 5C). These data showed that fluorescent reporters mirror the activity of endogenous promoters.