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Preparation and square wave electroporation of retinal explant cultures

Nature Protocols volume 1, pages 2710–2718 (2006)Cite this article

Abstract

This protocol details organotypic cultures of developing mouse, monkey and human retinas, which can be maintained for up to 2 weeks. Intact retinas are placed on polycarbonate filters floating on explant culture medium and fed every day with previously prepared retinal conditioned medium. Developing mouse retinas from E12.5 to P12 have been successfully cultured using this protocol as well as retinas from the equivalent stages of human and monkey development. Although this protocol does not require any special equipment, it provides a relatively high throughput. Retinal explant cultures lend themselves to complex pharmacological and genetic manipulations that are currently not feasible in vivo. A detailed procedure for square wave electroporation of retinal explants is also included to provide a high-throughput means to alter gene expression in the developing retina. This protocol for the preparation of retinal conditioned explant medium requires 4 d. Other steps of this protocol can be completed in 2 h.

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Figure 1: Retinal dissection and explant cultures.
Figure 2: Flowchart of the protocol showing three experimental designs.
Figure 3: Square wave electroporation for explant culture experiments.
Figure 4: In vivo square wave electroporation.

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Acknowledgements

This work was supported by grants (to M.A.D.) from the National Institutes of Health, Cancer Center Support from the National Cancer Institute, the American Cancer Society, Research to Prevent Blindness, the Pearle Vision Foundation, the International Retinal Research Foundation and the American Lebanese Syrian Associated Charities (ALSAC). M.A.D. is a Pew Scholar. This work was also supported by grants (to S.L.D.) from Fight for Sight and the Gerwin Fellowship Foundation.

Author information

Authors and Affiliations

  1. Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, 38105, Tennessee, USA

    Stacy L Donovan & Michael A Dyer

  2. Department of Ophthalmology, College of Medicine, University of Tennessee Health Science Center, Memphis, 38105, Tennessee, USA

    Michael A Dyer

Authors
  1. Stacy L Donovan
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Correspondence to Michael A Dyer.

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Supplementary information

Supplementary Video 1

Dissection of retinae from 6 P0 pups. (MOV 25438 kb)

Supplementary Video 2

Transfer immediately after dissection 3–4 dissected retinae with a small volume (50 μl) of culture medium to a membrane on the 12-well dish (1 retina per membrane) using a sterile disposable transfer pipette. (MOV 4650 kb)

Supplementary Video 3

Electroporation of dissected retina in the Petri dish electrode chamber. Transfer retina using a sterile disposable transfer pipette. (MOV 2781 kb)

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Donovan, S., Dyer, M. Preparation and square wave electroporation of retinal explant cultures. Nat Protoc 1, 2710–2718 (2006). https://doi.org/10.1038/nprot.2006.454

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