Neonatal Pial Surface Electroporation

Summary

The pial surface is a unique progenitor zone in the CNS that is receiving increasing attention. Herein, we detail a method for rapid genetic manipulation of this progenitor zone using a modified electroporation method. This procedure can be used for cellular and molecular investigations of cell lineages and signaling pathways involved in cell differentiation and to elucidate the fate and properties of daughter cells.

Abstract

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.

Introduction

Neural stem and progenitor cells are present throughout the mammalian CNS^1,2. Their nature and properties in embryonic and adult germinal zones surrounding the ventricular regions of the brain and spinal cord have been extensively documented in the past decade^1-3. In large part, this has been due to the development of increasingly precise genetic tools, such as nervous system specific Cre recombination of floxed alleles or retroviral lineage tracing⁴. However, one progenitor region—the pial surface progenitor zone—has only recently been described in any detail^5-7 and awaits comprehensiv....

Protocol

This procedure is in accordance with Cedars-Sinai IACUC requirements. Investigators should ensure institutional IACUC compliance prior to proceeding. All tools and reagents should be sterilized prior to use.

1. Preparation of Tools, Solutions, and DNA Mixture

1. Insert 100-mm fire polished borosilicate glass capillary tubes into a micropipette puller. Set heating to allow for standard weighted pull to form pipette tip of approximately 17.5 mm. Cut tips with sharp surgical scissors at a distance approximately 8-9 mm from the beginning of the tip to create roughly a 100 µm diameter opening.
2. Make a stock 1% w/v fast gre....

Results

Pial surface electroporation results in the expression of plasmid DNA in cells—mostly progenitors—at or near the pial surface⁶. More specifically, the orientation of the electrodes is critical in dictating the direction of plasmid movement and subsequent expression. Thus, in dual electrode configurations, the plasmid is directed in roughly a straight vector between the negative and positive electrode. Therefore, if the negative pole is placed over the injection site and the positive pole is ventral to the nega.......

Discussion

The most critical aspect for successful electroporation of pial surface progenitors are: 1) targeting of plasmid mix to the pial surface; 2) avoiding the generation of hematomas at the injection site; and 3) avoiding mortality associated with midbrain electroporation.

Appropriately targeting the pial surface is accomplished by measured and careful puncturing of the skull to avoid penetration of the pial surface. Improper targeting to the overlying skin or underlying ventricle or brain parenchy.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fire Polished Borosilicate Tubing	World Precision Instruments, Inc.	1B100F-4
Micropipette Puller	Sutter Instruments Company	P-30
Fast Green FCF	Sigma Aldrich, Inc.	F7528
XenoWorks Digital Microinjector	Sutter Instruments Company
ECM 830 Generator	Harvard Apparatus, BTX Instrument Div	45-0052
3-mm Platinum Tweezertrodes	Harvard Apparatus, BTX Instrument Div	45-0487
SignaGel Electrode Gel	Cardinal Health	70315-025
Tris-EDTA Buffer, pH 8.0	Integrated DNA Technologies, Inc.	11-01-02-05
Infrared Heat Lamp	VWR	36547-009
Fine Scissors Sharp	Fine Science Tools	14060-09