In vivo Electroporation of Developing Mouse Retina

Summary

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

Abstract

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.

We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane ^1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer ⁵. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein ^{6, 7, 8, 9, 10}.

DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate.

Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression ¹¹. Potential experiments are limited only by construct availability.

Protocol

1. Plasmid preparation for electroporation

The DNA concentration required for electroporation is 5μg/μl. This typically requires the desired plasmids to be amplified using a Maxi-prep (Qiagen) or equivalent method followed by a purification and concentration of the DNA to the working amount. The following steps describe the preparation of DNA to the working amount.

1. Aliquot 100 μg of DNA (from Maxi-prep or equivalent) and dilute the volume to 100 μL for ease of manipulation.
2. Add phenol; the amount of phenol should be calculated to obtain an approximately 60% DNA/volume ratio (μg DNA:total volume) i.e.: 100 μg DNA (in 1....

Discussion

In vivo electroporation represents a rapid and efficient method for the transformation of retinal cells with DNA expression plasmids. This method allows the experimenter to perform gain of function studies by ectopically introducing a gene of interest under the control of a ubiquitously expressed promoter or to perform loss of function studies by using shRNA constructs targeting genes of interest. Furthermore, multiple DNA plasmids can be electroporated simultaneously, allowing the experimenter to analyze multip.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Buffer Saturated Phenol	Invitrogen	15513-039	N/A
Chloroform	JT Baker	9180-03	N/A
Sodium Acetate	JT Baker	3470-05	3 M stock
Fast Green FCF	Fisher Scientific	BP123-10	10 % stock
Isopropyl alcohol prep	Tyco Healthcare, Covidien	6918	N/A
30-guage needle	Terumo Medical Corp.	SG2-3013	N/A
Exmire microsyringe	Ito Corporation	MS*E05	N/A
Tweezertrode (tweezer electrode)	BTX Technologies	522	N/A
Electro Square Porator (electroporator)	BTX Technologies	ECM 830	N/A
O.C.T. Compound	Sakura Finetek	4583	N/A