Name: induction of protein deletion through in utero electroporation to define deficits in neuronal migration in transgenic models
Uploaded: 2015-01-12T12:30:00
Description: Watch this Scientific Journal Video about Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models at JoVE.com

We are indebted to Pierre Mattar and Freda Miller for their assistance with the in utero electroporation and to Noel Ghanem and Bensun Fong for creating diagrams. We thank Linda Jui and Jason G. MacLaurin for excellent technical assistance. Equipment was supported by the Centre for Stroke Recovery.
This work was supported by a scholarship from HSFO and OGS to D.S.S. and a CIHR grant to R.S.S.

NOTE: All experiments were approved by the University of Ottawa&#8217;s Animal Care ethics committee, adhering to the Guidelines of the Canadian Council on Animal Care.
1. Pre-surgical Preparation and Materials
<ol>
	<li>Plasmid Preparation:
	<ol>
		<li>Prepare plasmids using the Qiagen MegaPrep according to the manufacturer&#8217;s protocol. Innoculate at least 400 ml of LB broth with transformed bacteria to ensure a high DNA yield and incubate overnight.</li>
		<li>Resuspend plasmid in 100...

<ol>
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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 &#181;l of fluid to appear following a pump of the foot paddle (Figure 1J). If the needle is too dull ...

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 molecules1-3. Since disruption of any of these mechanisms can impair neuronal migration in a transgenic mouse model4-8, determining the underlying cause of a defect in migration can be a complex and difficult process. In utero electroporation (IUE) can be used to complement and simplify interpretation of the phenotype of a genetic knock-out model and thereby elucidate important mechanisms required for radial migration7,9-11.
In utero electroporation (IUE) is the process by which a plasmid carrying both a gene of interest and a reporter is injected into the ventricle in the brain of a mouse or rat embryo and then drawn into the cells lining the ventricle through use of an electric current12-14. This allows the investigator to analyze the effect of up or down regulation of a gene of interest in development or function of electroporated neurons. Following IUE, brains can be processed for immunohistochemistry 7,9-11, electrophysiology 15 or cell culture 16,17. The major advantage of IUE is that is allows for highly specific manipulation of gene expression. Furthermore, IUE can be used to target a specific region of the developing brain through a directed current (Figure 2). IUE can also be used to target a specific cell type through injection of plasmids carrying genes under control of various promoters or plasmid activation systems (tetracycline induced gene expression is an example) 10,18-21.
IUE can be used in conjunction with the Cre-Lox mediated gene excision system 7-9,11,22. A plasmid containing a gene encoding the message for the Cre protein can be electroporated into an embryo homozygous for the floxed allele (in which the gene or specific DNA regions are flanked by two loxP sites for Cre mediated DNA recombination). Cre will then induce recombination of the gene of interest specifically in electroporated neural precursors, generating a knock-out of the floxed allele.The effect of protein knockdown on neural migration and development within individual neurons can then be studied. Electroporation of Cre induces recombination in only a small population of affected cells, leaving the supporting environment intact. In contrast, tissue specific expression of Cre under control of a cell type specific promoter, occurs throughout the entire tissue so that both migrating neuroblasts and the surrounding environment could be affected. Thus, juxtaposition of these two approaches can determine whether a given migration defect is due to cell autonomous or cell non-autonomous mechanisms. Defective migration in both experimental systems suggests that the observed phenotype results from a cell autonomous mechanism; normal migration following electroporation of Cre with defective migration in a tissue specific Cre model indicates that the gene of interest is acting through a non-cell autonomous mechanism.
IUE can also be used to perform rescue experiments by electroporating potential interacting genes into knock out animals 7,9,10. For example, an investigator could attempt to rescue a migration phenotype in a transgenic model by electroporating a downstream target and determining if the migration defect is corrected in the electroporated neurons. This has the additional benefit that a successful rescue indicates that normal migration can be restored by manipulating protein expression in a specific neuron, even though the surrounding environment is still deficient for the targeted gene. Again, this approach is more time and cost effective than crossing or generating transgenic lines, with the added benefit of determining whether the defective mechanism is cell autonomous.
IUE can be used to track migrating neurons through electroporation of a reporter plasmid into knock out embryos (Figure 4C). As only the neurons lining the ventricle at the time of surgery are electroporated 17, IUE can be used to follow the neurons born at a specific time with the advantage of visualization of their morphology in vivo.
Finally, it is possible to use IUE to target brain regions such as the medial or dorsal telencephalon, hippocampus or ganglionic eminence (Figure 2). This gives researchers the power to investigate gene function in a specific area independent of complications resulting from protein knockdown in neighboring structures.
Retinoblastoma protein (pRb), p107 and p130 comprise the pocket protein family and are well established regulators of cell cycle exit. However, there is increasing evidence that these proteins also regulate cell cycle independent aspects of neural development. As we have previously shown, pRb and p107 play a crucial role in both tangential 4,23 and radial migration 6. Here, we demonstrate the role of pocket protein family members pRb and p107 on neural migration 6 to exemplify the use of in utero electroporation of Cre in a transgenic model. In summary, IUE provides a powerful way of analyzing cell autonomous effects of gene deletion (or overexpression). When combined with tissue specific knock out models, IUE can provide additional information regarding the mechanisms controlling neural migration.

<table border="0" cellpadding="0" cellspacing="0" width="538">
	<tbody>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:233px;">Table 1: Equipment and Materials</td>
			<td style="width:64px;"></td>
			<td style="width:64px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:119px;">Product Name</td>
			<td style="width:115px;">Company</td>
			<td colspan="2" style="width:128px;">Catalog Number</td>
			<td style="width:177px;">Description</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:119px;">ECM 830 Square Wave Electroporator</td>
			<td style="width:115px;">BTX Harvard Apparatus</td>
			<td colspan="2" style="width:128px;">45-0052</td>
			<td style="width:177px;">Electroporator only &#8211; buy cables separately (45-0204)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">1250FS Footswitch for ECM 830</td>
			<td style="width:115px;">BTX Harvard Apparatus</td>
			<td colspan="2" style="width:128px;">45-0211</td>
			<td style="width:177px;">Foot paddle is necessary for hands-free electroporation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Tweezertrodes</td>
			<td style="width:115px;">BTX Harvard Apparatus</td>
			<td colspan="2" style="width:128px;">45-0489 (5mm)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 45-0488 (7mm)</td>
			<td style="width:177px;">Platinum forcep style electrodes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Femtojet</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920010504</td>
			<td style="width:177px;">Microinjector</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Griphead 0</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920007414</td>
			<td style="width:177px;">Holds the pulled needle</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Universal Capillary Holder</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920007392</td>
			<td style="width:177px;">Connects griphead to tube</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Injection Tube</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920007431</td>
			<td style="width:177px;">Connects capillary holder to Femtojet</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Foot Control</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920005098</td>
			<td style="width:177px;">Foot paddle for hands-free injection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Microloader Tips</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">930001007</td>
			<td style="width:177px;">Tips for loading the pulled capillary tubes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Plasmid Mega Kit</td>
			<td style="width:115px;">Qiagen</td>
			<td colspan="2" style="width:128px;">12181</td>
			<td style="width:177px;">5 QIAGEN-tip 2500, Reagents, Buffers</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Fast Green FCF</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td colspan="2" style="width:128px;">F7252-5G</td>
			<td style="width:177px;">Non-toxic dye</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Extra Fine Bonn Scissors</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">14084-08</td>
			<td style="width:177px;">For trimming the needles</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Bonn Strabismus Scissors</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">14084-09</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Graefe Forceps</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">11050-10</td>
			<td style="width:177px;">Straight</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Colibri Retractors</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">17000-03</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Stapler</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">12031-07</td>
			<td style="width:177px;">For 7mm clips</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Castroviejo Needle Holder</td>
			<td style="width:115px;">Medical Tools</td>
			<td colspan="2" style="width:128px;">SNH-6737</td>
			<td style="width:177px;">Straight, with lock</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Needle Puller</td>
			<td style="width:115px;">Narishige</td>
			<td colspan="2" style="width:128px;">PC-10</td>
			<td style="width:177px;">For pulling microcappillaries</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Microcapillaries</td>
			<td style="width:115px;">Drummond</td>
			<td colspan="2" style="width:128px;">1-000-800</td>
			<td style="width:177px;">0.4 mm inner diameter glass capillary tubes</td>
		</tr>
	</tbody>
</table>

induction of protein deletion through in utero electroporation to define deficits in neuronal migration in transgenic models

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&#8217;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.

Different regions of the brain can be targeted for electroporation by varying the orientation of the paddle electrodes over the embryo&#8217;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 (...

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

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.

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

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 ...

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