Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Summary

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

Abstract

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols¹. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Introduction

Dissociated cell cultures from embryonic chick retinas have been widely used to study various aspects of photoreceptor cell biology, including their survival^2-9, differentiation^10-12, neurite outgrowth¹³, and more. The advantages of this system, developed in the 1980s by Ruben Adler and collaborators and perfected by his and other groups^14-20, reside in the intrinsic characteristics of the chick as an animal model²¹. The large size of the chick eye, even at embryonic stages, provides large amounts of material for cultures. Moreover, when cultures are performed using embryonic day (ED) 5 - 6 retinas, 55 - 80% of th....

Protocol

All procedures described in this work were performed according to the guidelines recommended by the Animal Care and Use Committee at Johns Hopkins University.

1. Ahead of Time: Preparation of Instruments, Reagents and Dishes

1. Preparation of Eggs: Incubate fertilized White Leghorn chicken eggs at 37.5 °C and 60% relative humidity for 5 days in an egg incubator.
   NOTE: An incubator with rocking capacity may be helpful for standardizing and synchronizing embryonic development, but rocking is not strictly necessary for the outcome of this protocol.
2. Plasmid Preparation:
   1. Design plasmid construct for gen....

Results

Here we present a simple protocol for plasmid transfection into enucleated chick retinal cups for subsequent dissociated cell culture. Transfection is achieved by electroporation using easy to make custom electrodes (Figures 1 - 2). The parameters described in this protocol have been optimized to obtain transfection efficiencies that range between 20 and 27% (with an average of 22%) (Figure 3D). Notice that, for the reasons stated above, these results are quantified after 4 days in cultu.......

Discussion

The most critical step for the success of this protocol is selecting the appropriate stage of the embryos. In previous publications, a range of embryonic stages is given for these cultures, typically defined by the days of incubation or embryonic days (ED); thus it is usually assumed that using ED 5 to ED 6 embryos will yield equivalent results. However we have found that on stage 27 (ED 5), transfection efficiency will be around 22% of the total cell population, as stated above; yet efficiency will decrease to 16% if us.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ECM 830 Electro Square Porator	BTX/ Harvard Apparatus	45-0052
Genetrode, L-Shaped, 5 mm Gold Tip	BTX/ Harvard Apparatus	45-0115 model 512	Gold tipped electrode used as anode
Polyimide Tubing	Vention Medical	custom made	Internal Diameter: 0.5 mm/wall thickness: 0.15 - 0.2 mm. Used for insulating gold tiped electrode
2.5 mm square box filament, 4.5 mm wide	Sutter Instrument Company	FB245B	Used to make cathode electrode
HBSS, no calcium, no magnesium, no phenol red	Gibco - Life Technologies	14175-095
Moloney forceps	Roboz	RS-8254	Serrated; Slight Curve; 4.5" Length
Dumont tweezers 5/45	Roboz	RS-5058	Pattern #5, 45 Degree Angle; .10 X .06mm Tip Size; 109mm Length
Bonn micro forceps, 1x2 teeth	Roboz	RS-5172	Tying Platform; 1x2 Teeth, 0.12 mm Teeth; 3.75" Length; 0.3 mm Tip Width