Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments

Podsumowanie

This protocol describes a method to dissect, experimentally manipulate and culture whole retinal explants from chicken embryos. The explant cultures are useful when high success rate, efficacy and reproducibility are needed to test the effects of plasmids for electroporation and/or reagent substances, i.e., enzymatic inhibitors.

Streszczenie

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.

Wprowadzenie

The retina is part of the central nervous system and it is, with its relative simplicity and well-characterized cellular architecture, a popular model for studying central nervous system development. The eye of the chicken embryo is relatively large in comparison to the rest of the embryo. It is therefore easily accessible in ovo for experimental manipulations, such as injections or electroporation, and serves as an excellent tool to gain knowledge about retinal cell and developmental biology in vivo. Despite these major advantages, survival of the embryos can be low when experiments are invasive such as with electroporations, repeated injections, or combined experimental manipulations.

Electroporation of DNA plasmids into the chicken embryo in ovo is an important and well-established technique¹. It allows for labeling of neurons, tracing of cell fate as well as neuronal tracts in the central nervous system and it allows for ectopic gene expression to analyze protein function in vivo. The technique has been used for studies of neural tube², hindbrain³, and retina⁴. Electroporation of embryonic retina in ovo has some experimental difficulties that are related to the in vivo situation. The position of the eye, due to the cranial folding of the embryo, is relatively close to the heart. This proximity increases the risk of cardiac arrest following electroporation, and the risk increases with the age of the embryo. Moreover, to access the eye, it is necessary to open the embryonic membranes, thereby increasing the risk for bleeding, malformations and subsequent reduced viability. When testing and optimizing a new DNA plasmid often without a known phenotypic outcome, these limitations may decrease the efficacy and power of the method even for an experienced experimentalist. As presented in this protocol, the culture of the whole retinal explant, defined as the whole neural retina with the pigment epithelium removed, is an efficient method that complements the in ovo approach.

Intraocular injections of chemical reagents are relatively easy to perform in ovo. However, the effective and exact concentration of injected reagents within the neural retina may be difficult to fully control. The injected volume may vary due to leakage and the exact site of injection may affect both the distribution of the reagent within the eye and the diffusion through the vitreous body. The variability will have major implications for the interpretation of the results when i.e., a dose response curve for an enzyme inhibitor is determined; particularly if the effect is small and the temporal window of the effect is narrow. Moreover, only a single eye can be used from each embryo when performing in ovo experiments due to potential systemic effects via the blood stream onto the contralateral eye. Age matching is important when studying development and the individual variability between treated and control embryos may lead to additional experimental variability.

For these reasons, an ex ovo method based on retinal explants from chicken embryos was developed, in which the neural retina can be exposed to a uniform and controlled experimental condition in vitro. The present protocol was developed based on previous protocols^5-9. Retinal explants from stage (st) 20 (embryonic days [E] 3) to st31 (E7) chicken embryos were dissected, cultured and electroporated with a defined DNA plasmid concentration or exposed to a medium containing a defined concentration of a chemical reagent. The protocol presented here has been successfully implemented in recent publications, using several different chemical reagents, including regulators of the DNA damage pathway, such as KU55933, SB 218078, and NSC 109555 ditosylate, and the cell cycle, such as Cdk1/2 inhibitor III^10,11.

Protokół

This protocol is performed in accordance with the recommendations in the “Guide for the Care and Use of Laboratory Animals of the Association for research in vision and ophthalmology”.

1. Egg Handling and Eye collection

1. Store white Leghorn eggs at 12-14 °C for no longer than 1 week. If available, use a refrigerated wine cooler to store the fertilized eggs.
   Note: Higher store temperatures lead to abnormal development of embryos, while lower temperatures increases mortality. Extended storage time increases both mortality and abnormal development.
2. Incubate the eggs in a 37.5 °C and 60% humidified egg incubator with gentle rocking at a frequency of 5 times per 24 hr.
3. Remove the incubated eggs from the egg incubator at the desired embryonic age.
   Note: The age of the chicken embryo is determined as the time of incubation and is denoted as embryonic days (E) or as stages (st), according to the series of developmental stages described by Hamburger and Hamilton¹².
4. Place the selected eggs in the laminar flow hood. Wipe the eggshell with 70% ethanol to avoid contamination of the embryo.
   Note: Conduct all procedures under aseptic conditions with sterile solutions https://www.jove.com/science-education/5030/making-solutions-in-the-laboratory"lutions and instruments.
5. Tap the side of the egg firmly against a hard surface to crack open the egg. Place the content in a 100 mm Petri dish. Use a small spoon to transfer the embryo to a 35 mm Petri dish containing pre-warmed (37 °C) 1x PBS to prevent the tissue from drying.
6. Place the 35 mm Petri dish containing the embryonic tissue onto a binocular stereovision dissecting microscope in the laminar flow hood. Decapitate the embryo by pinching the neck with fine forceps and discard the body.
7. Use fine forceps to make an incision through the mouth, ventral to the eye. Starting at the site of incision, tear open the tissue surrounding the entire eye. Pinch off the optic nerve and remove the intact eye from the eye socket. Collect both the left and right eye from the same embryo for use as either the control or the treated eye.
8. Use a small spoon to transfer the eyes to a new 35 mm Petri dish containing pre-warmed (37 °C) 1x PBS.

2. Preparation of Whole Retinal Explants

1. Use fine forceps to remove all tissue around the eye including the scleral anlage.
   Note: The tissue will detach easily leaving the retina on the vitreous body and lens with the pigment epithelium intact (Figure 1A-B).
2. Use fine forceps to pinch a small incision into the pigment epithelium at the dorsal part of the eye without damaging the neural retina (Figure 1C).
3. Use fine forceps to gently tear open the pigment epithelium starting at the site of incision, leaving the lens and the entire retina attached to the vitreous body (Figure 1D). Keep the eye in warm (37 °C) 1x PBS to facilitate the removal of the pigment epithelium.
   Note: The pigment epithelium may be difficult to remove, in particular along the ciliary body around the pupil, in the anterior region of the eye, and along the choroid fissure. Therefore, some of the pigment epithelium may be left on the neural retina (Figure 1E-F).

3. Treatment of Whole Retinal Explants

1. Electroporation of whole retinal explants
   Note: Electroporation of whole retinal explants can be performed directly after step 2.
   1. Prepare culture medium containing 1:1 DMEM:F12 Nutrient mix, 10% FBS, 10 U/ml penicillin streptomycin, 5 µg/ml Insulin, and 2 mM L-glutamine.
   2. Cut off a 1.5 ml disposable plastic cuvette (12.5 mm x 12.5 mm x 45 mm) (or any small container capable of holding 300-400 µl solution) approximately 8 mm from the bottom using a fine-bladed saw.
   3. Dilute the DNA plasmid of choice in 1xDPBS to a final concentration of 0.1 µg/µl.
   4. Turn on the electroporator and set parameters to 15 V for st20 – st25 (E3-E4) retina and 20 V for st26 – st27 (E5) retina. Set the pulse-repeats to 5 pulses of 50 msec pulse-length with 1 sec intervals. Use a pulse generator that can be controlled by a foot-pedal on the floor as both hands will be needed to hold the electrodes in place.
   5. Connect two paddle shaped (flat, circular diam. 4 mm) platinum electrodes (Figure 2A) to the output socket on the pulse generator.
      Note: The platinum electrodes used in this protocol were made by the university workshop. The electrodes were made from 0.1 mm platinum plate “rondelles” (platinum grade: 950 Pt/5 Cu). The connecting 0.8 mm wire was insulated using plastic tubing.
   6. Carefully transfer the whole retinal explant from step 2.3 to the cuvette (or equivalent small container) using a polyethylene Pasteur pipette with the tip cut off to acquire the right tip-opening size for the retinal explant. Avoid using pipette tips as the retina tends to attach to the plastic and tear apart.
   7. Use a 100 µl pipette to gently remove the entire 1x PBS surrounding the retinal explant taking care not to touch the retina to avoid tearing.
   8. Add 100 µl plasmid solution to the cuvette to cover the whole retinal explant. Gently push down the retinal explant with forceps if it floats to the top. Use forceps or electrodes to position the lens to face any one side of the cuvette and the optic nerve to the bottom of the cuvette.
   9. Place the positive electrode in front of the lens and the negative electrode behind the retina (Figure 2A), without touching the tissue.
   10. Apply the current by pressing down the foot-pedal. Check for bubbles at the electrodes indicating successful discharge.
   11. Use a 100 µl pipette to remove all the plasmid mix from the cuvette without damaging the retina. The plasmid mix can be reused three to five times.
   12. Fill up the cuvette with pre-warmed (37 °C) 1x PBS. Use forceps to gently detach the retinal explant from the wall of the cuvette. After electroporation the retina is sometimes covered with bubbles, which makes it adhere to the wall of the cuvette.
   13. Use the polyethylene Pasteur pipette to transfer the whole retinal explant into a 24-well plate containing 1 ml pre-warmed (37 °C) culture medium per well. Incubate one retinal explant per well.
   14. Incubate the retinal explant at 37 °C on a rotator shaker, with a constant speed of 50 rpm, inside a cell incubator with 5% CO₂ for 24 hr. The rotation is to ensure maximum exposure to the medium and to prevent adhesion of the retina to the bottom of the 24-well plate.
   15. Proceed to step 4.
2. Treatment of whole retinal explants with chemical reagents
   Note: Chemical treatment of whole retinal explants can be performed directly after step 2.
   1. Prepare culture medium containing 1:1 DMEM:F12 Nutrient mix, 10% FBS, 10 U/ml penicillin streptomycin, 5 µg/ml Insulin, and 2 mM L-glutamine.
   2. Use a small spoon to transfer the retinal explant from step 2.3 into a 24-well plate containing 1 ml pre-warmed (37 °C) culture medium per well. Incubate one retinal explant per well.
   3. Incubate the whole retinal explant for 60 min at 37 °C on a rotator shaker, with a constant speed of 50 rpm, inside a cell incubator with 5% CO₂ before adding a chemical reagent or vehicle.
   4. Prepare the chemical solution, for example Cdk1/2 inhibitor III, an inhibitor of M-phase progression¹³, at a final concentration of 30 µM in DMSO.
   5. Remove the 24-well plate, containing the whole retinal explant, from the cell incubator. Add 10 µl of the chemical reagent or the vehicle (DMSO) directly to the retinal medium, resulting in a final concentration of 300 nM Cdk1/2 inhibitor III in 0.01% DMSO. Manually rotate the 24-well plate to allow for even distribution of the chemical reagent or vehicle in the solution.
   6. Incubate the retinal explants at 37 °C on a rotator shaker, with a constant speed of 50 rpm, inside an incubator with 5% CO₂ for the desired time (up to 24 hr).
   7. Proceed to step 4.

4. Fixation and Freezing of Whole Retinal Eexplants

1. Remove the 24-well plate containing the treated whole retinal explant from the cell incubator.
2. Use a small spoon to transfer the retinal explant to a 24-well plate containing 1 ml/well ice cold 4% paraformaldehyde in 1xPBS. Incubate on ice for 15 min.
3. Use a small spoon to transfer the whole retinal explant to a 24-well plate containing 1 ml/well ice cold 1xPBS to wash the retina. Incubate on ice for 10 min.
4. Use a small spoon to transfer the whole retinal explant to a 24-well plate containing 1 ml/well ice cold 30% sucrose in 1xPBS. Incubate on ice for 1-3 hr depending on the embryonic day of the retina. For st20 – st25 (E3) retinas, incubate for 1 hr, older retinas need longer incubation time in sucrose.
5. Use a small spoon to transfer the whole retinal explant onto a paraffin film containing approximately 500 µl cryostat freezing mounting medium. Remove as much sucrose as possible by gently moving the retinal explant in the freezing medium using the small spoon.
6. Transfer the retinal explant to an embedding mold containing freezing medium. Position the eye to facilitate future sectioning. Put the embedding mold containing the whole retinal explant on dry ice until the medium is frozen solid. Store at -80 °C.

Wyniki

This protocol describes the preparation (Figure 1A-F) and culturing of whole retinal explants from chicken embryos. This protocol has been successfully used for whole retinal explants from embryos of st20 (E3) to st31 (E7).

Electroporation of DNA plasmids into whole retinal explants allows for labeling and tracing of retinal progenitor cells or over-expression of different gene products. For electroporation experiments, the pigment epithelium was carefully removed from the enu...

Dyskusje

In this work a detailed protocol for dissection, electroporation or chemical treatment, and culturing of whole retinal explants from chicken embryos is presented. This protocol is easy, quick and allows for both a high success rate and reproducible results.

Electroporation of whole retinal explants produces large areas of cells that express the gene construct of interest. It is easy to correctly position the electrodes and to expose a specific portion of the retina to a define...

Materiały

Name	Company	Catalog Number	Comments
1xPBS (tablet)	Life technologies	18912-014
10x DPBS	Life technologies	14080-048
100 mm Petri dish	VWR	734-0006
100 μl pipette tips	VWR	613-0798
1.5 ml disposable plastic cuvette	Thomas Scientific	8495V01
24-well plate	Sigma Aldrich	D7039
35 mm Petri dish	VWR	391-1998
70% ethanol	Solveco	1054
Cdk1/2 inhibitor III	217714	Calbiochem	300 nM in 0.01% DMSO
Cell culture incubator	Thermo Forma
Dissecting microscope	Leica
DMEM	Life Technologies	41966-029
Electrodes			Platina, custom made
Electro square porator ECM 830	Harvard Apparatus
F12 Nutrient mix	Life Technologies	31331-028
FBS	Life Technologies	16140-071
Forceps	AgnThos	0108-5-PS
Freezing medium NEG50	Cellab, Sweden	6502
GFP expressing DNA plasmid (pZGs)
Humidified incubator	Grumbach Brutgeraete GmbH, Asslar, Germany	8204
Insulin	Sigma Aldrich	I9278-5ML
L-glutamine	Life Technologies	25030024
Mounting medium ProLong Gold with DAPI	Life Technologies	P36935
Paraffin film	VWR	291-1214P
Paraformaldehyde	Sigma Aldrich	16005-1KG-R
Peel-A-Way embedding mold	Sigma Aldrich	E6032
Penicillin streptomycin	Life Technologies	15140-122
PhosphoHistone 3 (PH3)	Millipore	06-570	Dilution 1/4,000
Platinum electrodes (custom made from "rondelles")	Sargenta	390-R (rondeller)	Dia: 4mm, 0.1 mm thickness
Platimun electrodes	Sonidel	CUY700P4L	Dia: 4 mm
Polyethylene pasteur pipette	VWR	612-2853
Rotator shaker	VWR	444-2900
Small spoon	VWR	231-2151
Sucrose	VWR	443815S
White Leghorn eggs			Local supplier
Wine cooler	WineMaster 24, Caso, Berlin Germany