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Efficient Gene Knockdown in Adult Zebrafish Retina by Intravitreal Injection

Eyad Shihabeddin,Abirami Santhanam,Stephan Tetenborg,Alexandra L. Aronowitz,John O’Brien

Published 2025 in Bio-protocol

ABSTRACT

High-throughput sequencing has created a tremendous amount of information about the genes expressed in various cell types and tissues throughout the body. As such, there is a need for a quick and effective method to knock down genes of interest in order to investigate their roles. While there are many approaches for this in mammalian models, there are limited ways to knock down genes of interest in adult zebrafish. Unlike mammals, zebrafish have the natural ability to regenerate their neurons after injury or disease is detected, making them a staple in regenerative studies. Unfortunately, current approaches for gene knockdown in the retina of adult zebrafish are costly and provide a barrier for many scientists. We provide two cost-effective approaches for targeted gene knockdowns in adult zebrafish retinas. We describe this approach through the use of Vivo-morpholinos and lipid-encapsulated siRNAs that target the expression of the proliferating cell nuclear antigen (PCNA) gene in adult zebrafish. We also describe how to collect and process retina samples for downstream immunohistochemistry, imaging, and quantification. Overall, this protocol will provide researchers with a straightforward, cheap, and effective method to perform targeted gene knockdowns in adult zebrafish retinas. Key features • This protocol provides researchers with an approach to knock down genes of interest in adult zebrafish retina without the use of electroporation. • This protocol can be performed without causing an acute damage response in the retina. • This protocol allows targeting of genes in both proliferating cells and terminally differentiated cells. • This protocol allows the retina to be collected and processed for further downstream analysis.

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