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Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Neuroscience.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Neuroscience.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Neuroscience.
2015 (English)In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, no 103, e53202Article in journal (Refereed) Published
Resource type
Text
Abstract [en]

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental manipulations in ovo/in vivo makes the chicken embryonic retina a versatile and very efficient experimental model. Although the chicken retina is easy to target in ovo by intraocular injections or electroporation, the effective and exact concentration of the reagents within the retina may be difficult to fully control. This may be due to variations of the exact injection site, leakage from the eye or uneven diffusion of the substances. Furthermore, the frequency of malformations and mortality after invasive manipulations such as electroporation is rather high. This protocol describes an ex ovo technique for culturing whole retinal explants from chicken embryos and provides a method for controlled exposure of the retina to reagents. The protocol describes how to dissect, experimentally manipulate, and culture whole retinal explants from chicken embryos. The explants can be cultured for approximately 24 hr and be subjected to different manipulations such as electroporation. The major advantages are that the experiment is not dependent on the survival of the embryo and that the concentration of the introduced reagent can be varied and controlled in order to determine and optimize the effective concentration. Furthermore, the technique is rapid, cheap and together with its high experimental success rate, it ensures reproducible results. It should be emphasized that it serves as an excellent complement to experiments performed in ovo.

Place, publisher, year, edition, pages
2015. no 103, e53202
Keyword [en]
Developmental Biology, Issue 103, Culture, explant, eye, inhibitors, in ovo, in vitro, plasmid
National Category
Neurosciences
Identifiers
URN: urn:nbn:se:uu:diva-268713DOI: 10.3791/53202ISI: 000364222300048OAI: oai:DiVA.org:uu-268713DiVA: diva2:878804
Funder
Swedish Childhood Cancer Foundation, PR2013-0104Swedish Research Council, 12187-18-3
Available from: 2015-12-09 Created: 2015-12-09 Last updated: 2017-12-01
In thesis
1. Keeping up with retinal photoreceptors and horizontal cells: Labelling and mapping of cells in the normal and diseased embryonic chicken retina
Open this publication in new window or tab >>Keeping up with retinal photoreceptors and horizontal cells: Labelling and mapping of cells in the normal and diseased embryonic chicken retina
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The childhood eye cancer retinoblastoma originates from the retina and its development is initiated while the foetus is in the uterus. Retinoblastoma has a reported incidence of 1 in 15-18 000 live births, and approximately 90% of all patients are diagnosed before the age of 5. The occurrence of retinoblastoma is usually detected by the parents and the most frequent symptoms are leukocoria (white pupillary reflex), strabismus (squinting) or if the child complains of visual problems. Retinoblastoma is diagnosed by examination under anaesthesia and documentation by RetCam. It is treated with various cytostatic agents, or by laser. If the treatment is unsuccessful, or there is a risk that the tumour cells will spread and form metastases, the eye is removed.

Previous studies have indicated that the cell type from which the tumour arises, the cell-of-origin, may be the cone photoreceptors and/or their immediate interneuron, the horizontal cells. Determining the cell-of-origin for retinoblastoma is an important goal, however, understanding the molecular mechanisms that distinguish the photoreceptors and the horizontal cells from the other retinal cells may prove just as important for understanding this disease.

The aim of my project has been to develop, optimise and validate methods to label, map and target expression to photoreceptors and horizontal cells in the chicken embryonic retina. We have successfully established several methods that test the expression pattern of conserved, regulatory DNA sequences, and have performed short- and long-term expression of various genes that have been reported to be involved in cell cycle regulation and cell fate determination. One of my most important findings was that a region from the RXRγ gene allowed us to specifically target the photoreceptors and horizontal cells. Our previous knowledge, together with the newly established tools, puts us an important step closer towards understanding the development and behaviour of the retinal photoreceptors and horizontal cells, however, further studies are of course needed.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. 62 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1302
Keyword
Chicken, electroporation, horizontal cells, photoreceptors, retina, piggyBac
National Category
Neurosciences
Identifiers
urn:nbn:se:uu:diva-315655 (URN)978-91-554-9825-2 (ISBN)
Public defence
2017-04-07, B21, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2017-03-10 Created: 2017-02-17 Last updated: 2017-03-20

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Blixt, MariaHallböök, Finn

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