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A universal protocol for isolating retinal ON bipolar cells across species via fluorescence-activated cell sorting - RNAseq of cell populations

ABSTRACT: A universal protocol for isolating retinal ON bipolar cells across species via fluorescence-activated cell sorting - RNAseq of cell populations

PROVIDER: PRJEB40891 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR4695810_1.fastq.gz	Fastqsanger.gz
	ERR4695810_2.fastq.gz	Fastqsanger.gz
	ERR4695811_1.fastq.gz	Fastqsanger.gz
	ERR4695811_2.fastq.gz	Fastqsanger.gz
	ERR4695802_1.fastq.gz	Fastqsanger.gz

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A universal protocol for isolating retinal ON bipolar cells across species via fluorescence-activated cell sorting - RNAseq of cell populations

Project description:Many inherited retinal dystrophies (IRDs) are characterized by progressive degeneration and loss of light-sensitive photoreceptors. The most promising treatment concept for IRDs is gene supplementation therapy with viral vectors, which relies on the presence of viable photoreceptors at the time of intervention. At later stages, when the pathology is too advanced, photoreceptors are lost and cannot be rescued. For late-stage patients, conferring light sensing abilities to the remaining ON circuit interneurons (i.e., ON bipolar cells) using optogenetic tools poses an alternative treatment approach. However, the development of such therapies is hampered by the lack of efficient gene delivery tools targeting ON bipolar cells, which in turn rely on effective cell isolation for tool development. In this work we have developed a pipeline to isolate ON bipolar cells by relying on the recognition of two intracellular markers and the isolation via fluorescence-activated cell sorting (FACS). As fixation proved to be a necessary prerequisite, RNA sequencing and real-time PCR were used not only to confirm the identity of the isolated ON bipolar cells but also to prove the compatibility of this method with highly sensitive techniques. Moreover, we have shown that this protocol can be applied without any substantial modification to the retinas of wild-type and Rd1 mutant mice as well as retinas of non-human primates (NHP).

2021-01-26 | E-MTAB-9685 | biostudies-arrayexpress

A universal protocol for isolating retinal ON bipolar cells across species via fluorescence-activated cell sorting.

Project description:Inherited retinal dystrophies (IRDs) are characterized by progressive degeneration and loss of light-sensing photoreceptors. The most promising therapeutic approach for IRDs is gene supplementation therapy using viral vectors, which requires the presence of viable photoreceptors at the time of intervention. At later disease stages, photoreceptors are lost and can no longer be rescued with this approach. For these patients, conferring light-sensing abilities to the remaining interneurons of the ON circuit (i.e., ON bipolar cells) using optogenetic tools poses an alternative treatment strategy. Such treatments, however, are hampered by the lack of efficient gene delivery tools targeting ON bipolar cells, which in turn rely on the effective isolation of these cells to facilitate tool development. Herein, we describe a method to selectively isolate ON bipolar cells via fluorescence-activated cell sorting (FACS), based on the expression of two intracellular markers. We show that the method is compatible with highly sensitive downstream analyses and suitable for the isolation of ON bipolar cells from healthy as well as degenerated mouse retinas. Moreover, we demonstrate that this approach works effectively using non-human primate (NHP) retinal tissue, thereby offering a reliable pipeline for universal screening strategies that do not require inter-species adaptations or transgenic animals.

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