Fig. 1: Vector design for insertion of a Tet-On regulated RGC cassette. | npj Regenerative Medicine

Fig. 1: Vector design for insertion of a Tet-On regulated RGC cassette.

From: Human retinal ganglion cell neurons generated by synchronous BMP inhibition and transcription factor mediated reprogramming

Fig. 1

a A conceptual diagram for a 3G Tet-ON system where doxycycline binds to constitutively expressed rtTA to induce polycistronic expression of NEUROG2, ATOH7, ISLET1, and POU4F2 (NAIP2) leading to conversion of RGC-like neurons. b Electroporation approach for the transient transfection of the NAIP2 transgene package. c PSCs that were transiently transfected with control (empty) or d NAIP2 plasmids and treated with dox for 3 days. Black arrows = PSCs, white arrows = converted neurons. Brightfield and fluorescent images of e the POU4F2-tdTomato+ reporter in a 66-day-old retinal organoid in the IMR90.4 genetic background and f a 65-day-old POU4F2-h2b-mNeonGreen+ retinal organoid in the WA09 genetic background. g U6 promoter-driven expression of an AsCas12a cRNA targeting the CLYBL safe harbor site for insertion of a zeocin selectable Tet-inducible transgene cassette. Scale c, d = 100 µm, e, f = 400 µm.

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