Understanding heterochromatin redistribution in mouse retina by designing and implementing a novel reporter cell-line in retinal organoids

Abstract

In eukaryotic cells, DNA is packed in the form of chromatin distributed three-dimensionally. This allows for the control of gene expression based on the location of chromatin. Euchromatin is transcriptionally active and located at the nuclear interior, while heterochromatin is transcriptionally inactive and found at the nuclear periphery. This is known to be the conventional organization and is found across vertebrates. However, mature nocturnal rod photoreceptors in the retina have the opposite nuclear organization. Hence, euchromatin is located at the nuclear periphery and heterochromatin at the nuclear interior. The purpose of this project is to optimize and develop tools to study the rod-specific nuclear organization.

The Kind-lab developed the scDam&T approach, which allows for the simultaneous measurement of genomic positioning and transcription in single cells. Combining this method with an mESC-based in vitro organoid system would allow us to study and perturb the inversion. However, preliminary data showed that the Dam-LaminB1 F1ES line could not differentiate into retinal organoids. First, I optimized the mouse retinal organoid protocol and tested multiple cell lines. Next, we generated an inducible IB10-based Dam-LaminB1 cell line. From this we were able to grow organoids which showed all the hallmarks of retinal organoids.