In ovo Expression of MicroRNA in Ventral Chick Midbrain

Summary

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

Abstract

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

Introduction

The recognition of small non-coding RNAs as additional players for gene expression launched a new complexity to genomic programming/gene regulation. Different species of non-coding RNAs have functional importance in neural cells, including small non-coding RNAs^1-4. MicroRNAs (miR or miRNA) for example show distinct and changing expression profiles in developing brains⁵. Targeted in ovo electroporation of chick embryos provides a unique opportunity for temporal and spatial control of gene expression and silencing during development.

This video demonstrates the different steps of performing ectopic expression of miRs in specific areas of the chick midbrain using in ovo electroporation^6-10. To ensure a long lasting effect of these small non-coding RNAs in cells, the DNA sequence of miRs were cloned into mono- or bi-cistronic vectors. For in ovo electroporation, miR containing vector is injected into the midbrain neural tube by exposing the embryo after making a small window in the egg shell. To transfect specific areas of the midbrain small plus (anode) and minus (cathode) platinum electrodes are placed at specific positions. For ventral midbrain transfection, the anode is placed underneath the left ventral midbrain and the cathode above the right half of the midbrain before applying a current. The opening in the eggshell is closed with tape and embryos are incubated for as long as required for any analysis. This method was originally described by Muramatsu et al.⁶ and improved by Momose et al.⁸ for specific area transfection.

Schematic Overview.

1. The embryo in the egg is exposed by cutting a small window into the eggshell.
2. The dissolved vector(s) is injected into the midbrain using a micro capillary.
3. Two electrodes – placed parallel or under and above the embryo – generate a pulsed electric field.
4. The electric field temporally creates pores in the cell membrane, which facilitate entry into the cell by the negatively charged DNA (or RNA) attracted to the anode^11,12.

Protocol

1. Requirements for In ovo Electroporation Preparing the vector construct: miR sense and antisense primers were designed after the instructions of Ambion, annealed and ligated into ApaI/EcoRI digested pSilencer U6.1 vector. After successful transformation one of the resulting clones was grown and pSilencer plasmid was isolated by alkaline lysis method using a Quiagen midi preparation kit. Electrodes: Electrodes can be purchased or easily be built<…

Representative Results

The extent of the midbrain area transfected with miR is visible through the expression of GFP from a reporter vector injected along with the miR expressing vector (Figure 2). Using platinum electrodes with a diameter of 0.5 mm and placed parallel to the AP axis of midbrain leads normally to the transfection of a broad area along the DV- and AP- axis, including hindbrain, midbrain and sometimes diencephalon (Figures 2A and B). The size of the electrodes results in a wide electric field ap…

Discussion

This video demonstrates an effective method to transfect plasmid into the neuroepithelial cells of specific areas of the chick midbrain. Rectangular electric pulses of low voltage can introduce DNA into cells of the chick neural tube in ovo^6,16. However, the accuracy of DNA targeting is often hindered by the wide electric field, which rises through the relatively large electrodes (Φ = 0.5 mm). We tried to tackle that problem by using electrodes smaller in diameter following the guidelines of Momos…

Materials

Name	Company		Model
Borosillicate glass capillaries	Hartenstein		Model: 0.9 mm
Microcapillary puller	WPI, Berlin		Model: Pul1-E
Electroporator	Intracel		Model: TSSIC
Stereomicroscope – fluorescence	LEICA		Model: MZFLIII
Stereomicroscope	Zeiss		Model: Stemi
Camera and software	Zeiss		Model: Axiocam MRc/ Axiovision Re. 4.8