Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Summary

Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment.

Abstract

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific Cre models, deletion of a protein throughout a tissue or cell population often leads to complex phenotypes resulting from multiple interacting mechanisms. Determining whether a phenotype results from disruption of a cell autonomous mechanism, which is intrinsic to the cell in question, or from a non-cell autonomous mechanism, which would result from impairment of that cell’s environment, can be difficult to discern. To gain insight into protein function in an in vivo context, in utero electroporation (IUE) enables gene deletion in a small subset of cells within the developing cortex or some other selected brain region. IUE can be used to target specific brain areas, including the dorsal telencephalon, medial telencephalon, hippocampus, or ganglionic eminence. This facilitates observation of the consequences of cell autonomous gene deletion in the context of a healthy environment. The goal of this protocol is to show how IUE can be used to analyze a defect in radial migration in a floxed transgenic mouse line, with an emphasis on distinguishing between the cell autonomous and non-cell autonomous effects of protein deletion. By comparing the phenotype resulting from gene deletion within the entire cortex versus IUE-mediated gene deletion in a limited cell population, greater insight into protein function in brain development can be obtained than by using either technique in isolation.

Introduction

Radial migration is a central process to early cortical development. It depends on a variety of cell autonomous factors, such as correct mitotic exit and neuronal differentiation, cell polarity, regulation of cytoskeletal dynamics and expression of transmembrane receptors, as well as non-cell autonomous factors, such as formation of the radial glial scaffold and secretion of migratory guidance molecules^1-3. Since disruption of any of these mechanisms can impair neuronal migration in a transgenic mouse model^4-8, determining the underlying cause of a defect in migration can be a complex and difficult process. In utero electroporation (IUE)....

Protocol

NOTE: All experiments were approved by the University of Ottawa’s Animal Care ethics committee, adhering to the Guidelines of the Canadian Council on Animal Care.

1. Pre-surgical Preparation and Materials

1. Plasmid Preparation:
   1. Prepare plasmids using the Qiagen MegaPrep according to the manufacturer’s protocol. Innoculate at least 400 ml of LB broth with transformed bacteria to ensure a high DNA yield and incubate overnight.
   2. Resuspend plasmid in 100-200 µl TE to a final concentration of at least 5 µg/µl. Aliquot DNA and store at -20 °C. If the concentration is too low, do not ....

Results

Different regions of the brain can be targeted for electroporation by varying the orientation of the paddle electrodes over the embryo’s head (Figure 2). For all parts of the forebrain, the plasmid is injected into the lateral ventricle (the hind brain can be targeted by injecting into the fourth ventricle). In general, place the electrodes so that the straight line between them passes through the region of interest, with DNA being drawn toward the positive electrode. To target the lateral cortex (

Discussion

Two of the primary challenges in performing in utero electroporations are 1) preventing embryo lethality and 2) decreasing variability between electroporations. One of the most important factors in preventing lethality is needle quality, as blunt needles inflict more damage to the embryo. When cutting the needle, keep the tip as long as possible while still allowing a visible droplet of 0.1-0.2 µl of fluid to appear following a pump of the foot paddle (Figure 1J). If the needle is too dull .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ECM 830 Square Wave Electroporator	BTX Harvard Apparatus	45-0052	Electroporator only – buy cables separately (45-0204)
1250FS Footswitch for ECM 830	BTX Harvard Apparatus	45-0211	Foot paddle is necessary for hands-free electroporation
Tweezertrodes	BTX Harvard Apparatus	45-0489 (5mm)         45-0488 (7mm)	Platinum forcep style electrodes
Femtojet	Eppendorf	920010504	Microinjector
Griphead 0	Eppendorf	920007414	Holds the pulled needle
Universal Capillary Holder	Eppendorf	920007392	Connects griphead to tube
Injection Tube	Eppendorf	920007431	Connects capillary holder to Femtojet
Foot Control	Eppendorf	920005098	Foot paddle for hands-free injection
Microloader Tips	Eppendorf	930001007	Tips for loading the pulled capillary tubes
Plasmid Mega Kit	Qiagen	12181	5 QIAGEN-tip 2500, Reagents, Buffers
Fast Green FCF	Sigma-Aldrich	F7252-5G	Non-toxic dye
Extra Fine Bonn Scissors	Fine Science Tools	14084-08	For trimming the needles
Bonn Strabismus Scissors	Fine Science Tools	14084-09
Graefe Forceps	Fine Science Tools	11050-10	Straight
Colibri Retractors	Fine Science Tools	17000-03
Stapler	Fine Science Tools	12031-07	For 7mm clips
Castroviejo Needle Holder	Medical Tools	SNH-6737	Straight, with lock
Needle Puller	Narishige	PC-10	For pulling microcappillaries
Microcapillaries	Drummond	1-000-800	0.4 mm inner diameter glass capillary tubes