Name: tracing gene expression through detection of &#946;-galactosidase activity in whole mouse embryos
Uploaded: 2018-06-26T14:00:00
Description: Watch this Scientific Journal Video about Tracing Gene Expression Through Detection of ÃŽÂ²-galactosidase Activity in Whole Mouse Embryos at JoVE.com

We would like to thank the Histopathological Service for their technical assistance at the Centro Nacional de Investigaciones Cardiovasculares (CNIC). We also thank Dr. Motoharu Seiki for kindly providing Mt4-mmpLacZ mice, and Dr. Alicia G. Arroyo for supporting our project and for her critical reading of the manuscript. We wish to thank Peter Bonney for proofreading this article. This work was supported by Universidad Europea de Madrid by means of a grant (# 2017UEM01) awarded to C.S.C.

All experimental procedures were approved by the Committee on the Ethics of Animal Experiments of the CNIC (Centro Nacional de Investigaciones Cardiovasculares) and the Comunidad Aut&#243;noma de Madrid to ensure minimal animal suffering.
1. Collection of Embryos from Pregnant Mice (from E8.5 to E12.5)
<ol>
	<li>Sacrifice pregnant mice by either cervical dislocation or CO2 inhalation. The day of the first observed vaginal plug was considered embryonic day 0.5 (E0.5).</li>
	<li>Lay...

The E. coli LacZ gene has been widely used as a reporter in studies of gene expression patterns because of its high sensitivity and ease of detection. The present protocol describes a classic method for detecting &#946;-gal expression based on an enzymatic reaction that is easy and quick to perform as well as inexpensive. This method can be also applied without major modifications in whole mount embryos, intact organs, cryostat tissue sections or cultured cells.
Accurate application o...

In order to describe specific gene expression patterns, the use of reporter genes as markers has been paramount from Drosophila to mammals. In experiments involving transgenic and knockout animals, the bacterial &#946;-galactosidase gene (LacZ) of Escherichia coli (E. coli) is one of the most widely used1,2,3,4. &#946;-galactosidase (&#946;-gal) catalyzes the hydrolysis of &#946;-galactosides (such as lactose) into its monosaccharides (glucose and galactose)5. Its most commonly used substrate is X-gal (5-bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside), a glycoside that is hydrolyzed by &#946;-galactosidase giving rise to 5-bromo-4-chloro-3-hydroxyindole and galactose. The first is oxidized into a dimer that, when used combined with potassium ferri-and ferro-cyanide, produces a characteristic insoluble, blue color precipitate (Figure 1)6.
The LacZ gene started to be used as a reporter gene over thirty years ago7,8. Usually, LacZ is inserted downstream of an endogenous promoter in the place of the open reading frame, so it can be used in bacterial and cell culture to visualize cells containing a particular insert, as well as in transgenic animals as a tracer of endogenous gene expression patterns during development9. In this regard, the visualization of &#946;-galactosidase activity has been extensively used in Drosophila to understand the developmental and cellular processes from single cells to whole tissues. Drosophila genetics favor the generation of stable lines in which a modified P-element construct containing the reporter gene LacZ is inserted at random locations in the genome. Thus, when placed under the influence of enhancer elements it may drive its expression in a tissue specific manner, which has allowed the systematic analysis of the expression patterns of many genes during the past two decades10. In addition, the use of transgenic mice to monitor LacZ gene expression also allows detection of gene recombination events by Cre-loxP mediated recombination, and localization of the mutant embryonic stem cell derivatives in chimeric analyses11, which facilitates the control of LacZ expression in specific tissues as well as temporally. Also, in whole embryos, detection of the &#946;-galactosidase activity may produce differential staining patterns at different intensities that can be conveniently observed across different developmental stages to analyze temporal changes in gene expression8,12.
In this article, we present a protocol to visualize gene expression through X-gal staining in the whole mount tissue at early developmental stages of mouse embryos. We present this histochemical method as a highly sensitive and inexpensive technique that favors accurate detection of the labeled cells either in whole mount specimens or at the cellular level after paraffin embedded tissues or embryos. The method allows for the direct visualization of staining in the mouse tissue with the minimum background when compared with other methods13.

<table>
	<tbody>
		<tr>
			<td>REAGENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>SIGMA-ALDRICH</td>
			<td>24137-1L-R</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>SCHARLAU</td>
			<td>50004/ LE3Q2014</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqueous mounting medium</td>
			<td>VECTOR LABS</td>
			<td>H-5501</td>
			<td></td>
		</tr>
		<tr>
			<td>Synthetic mounting media</td>
			<td>MERCK</td>
			<td>100579</td>
			<td></td>
		</tr>
		<tr>
			<td>96% Ethanol</td>
			<td>PROLABO</td>
			<td>20824365</td>
			<td></td>
		</tr>
		<tr>
			<td>99.9% Ethanol absolute</td>
			<td>SCHARLAU</td>
			<td>ET00021000</td>
			<td></td>
		</tr>
		<tr>
			<td>50% Glutaraldehyde solution</td>
			<td>SIGMA-ALDRICH</td>
			<td>G6403-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>85% Glycerol</td>
			<td>MERCK</td>
			<td>104094</td>
			<td></td>
		</tr>
		<tr>
			<td>99.9% Glycerol</td>
			<td>SIGMA-ALDRICH</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>SIGMA-ALDRICH</td>
			<td>63064</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonionic surfactant (Nonidet P-40)</td>
			<td>SIGMA-ALDRICH</td>
			<td>542334</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclear Fast Red counterstain</td>
			<td>SIGMA-ALDRICH</td>
			<td>N3020</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin pastilles</td>
			<td>MERCK</td>
			<td>111609</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>SIGMA-ALDRICH</td>
			<td>158127-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (tablets)</td>
			<td>SIGMA-ALDRICH</td>
			<td>P4417-50TAB</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium ferrocyanate</td>
			<td>MERCK</td>
			<td>1049840500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium ferrocyanide</td>
			<td>MERCK</td>
			<td>1049731000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>SIGMA-ALDRICH</td>
			<td>S8032</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>SIGMA-ALDRICH</td>
			<td>30970</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate monohydrate</td>
			<td>SIGMA-ALDRICH</td>
			<td>106346</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic dihydrate</td>
			<td>SIGMA-ALDRICH</td>
			<td>71638</td>
			<td></td>
		</tr>
		<tr>
			<td>Thymol</td>
			<td>SIGMA-ALDRICH</td>
			<td>T0501</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris hydrochloride (Tris HCl)</td>
			<td>SIGMA-ALDRICH</td>
			<td>10812846001 (Roche)</td>
			<td></td>
		</tr>
		<tr>
			<td>X-GAL</td>
			<td>VENN NOVA</td>
			<td>R-0004-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>VWR CHEMICALS</td>
			<td>VWRC28973.363</td>
			<td></td>
		</tr>
		<tr>
			<td>EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable plastic cryomolds 15x15x5 mm</td>
			<td>SAKURA</td>
			<td>4566</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotatory Microtome</td>
			<td>Leica</td>
			<td>RM2235</td>
			<td></td>
		</tr>
		<tr>
			<td>Cassettes</td>
			<td>Oxford Trade</td>
			<td>OT-10-9046</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Cover Glasses 24x60 mm</td>
			<td>VWR</td>
			<td>ECN631-1575</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Thermo Scientific, MENZEL-GL&#196;SER</td>
			<td>AGAA000001#12E</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesion microscope slides</td>
			<td>Thermo Scientific, MENZEL-GL&#196;SER</td>
			<td>J1820AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Flotation Water bath</td>
			<td>Leica</td>
			<td>HI1210</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Low Profile Microtome Blades</td>
			<td>Feather</td>
			<td>UDM-R35</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin oven</td>
			<td>J.R. SELECTA</td>
			<td>2000205</td>
			<td></td>
		</tr>
		<tr>
			<td>Wax Paraffin dispenser</td>
			<td>J.R. SELECTA</td>
			<td>4000490</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>DM500</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene microcentrifuge tubes 2.0 mL</td>
			<td>SIGMA-ALDRICH</td>
			<td>T2795</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene microcentrifuge tubes 1.5 mL</td>
			<td>SIGMA-ALDRICH</td>
			<td>T9661</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>IKA Labortechnik</td>
			<td>HS250 BASIC</td>
			<td></td>
		</tr>
		<tr>
			<td>Stirring Hot Plate</td>
			<td>Bibby</td>
			<td>HB502</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Shaker</td>
			<td>IKA Labortechnik</td>
			<td>MS1</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory scale</td>
			<td>GRAM</td>
			<td>FH-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision scale</td>
			<td>Sartorius</td>
			<td>ISO9001</td>
			<td></td>
		</tr>
		<tr>
			<td>pHmeter</td>
			<td>Crison</td>
			<td>Basic 20</td>
			<td></td>
		</tr>
		<tr>
			<td>Optic fiber</td>
			<td>Optech</td>
			<td>PL2000</td>
			<td></td>
		</tr>
	</tbody>
</table>

tracing gene expression through detection of &#946;-galactosidase activity in whole mouse embryos

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. The classical histochemical reaction is based on the hydrolysis of the substrate X-gal in combination with ferric and ferrous ions, which produces an insoluble blue precipitate that is easy to visualize. Therefore, &#946;-galactosidase activity serves as a marker for the expression pattern of the gene of interest as the development proceeds. Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the subsequent method for paraffin sectioning and counterstaining. Additionally, a procedure for clarifying whole embryos is provided to better visualize X-gal staining in deeper regions of the embryo. Consistent results are obtained by performing this procedure, although optimization of reaction conditions is needed to minimize background activity. Limitations in the assay should be also considered, particularly regarding the size of the embryo in whole mount staining. Our protocol provides a sensitive and a reliable method for &#946;-galactosidase detection during the mouse development that can be further applied to the cryostat sections as well as whole organs. Thus, the dynamic gene expression patterns throughout development can be easily analyzed by using this protocol in whole embryos, but also detailed expression at the cellular level can be assessed after paraffin sectioning.

Here we show the results from applying the standard protocol for the &#946;-galactosidase histochemical reaction using X-gal as the substrate in whole mouse embryos (Figure 1 and Figure 2). By using this protocol, we examine Membrane type 4-matrix metalloproteinase (Mt4-mmp) expression at different embryonic developmental stages (E9.5, E11.5, and E12.5) using Mt4-mmp mutant mice that express the LacZ reporter under the control of...

Watch this Scientific Journal Video about Tracing Gene Expression Through Detection of ÃŽÂ²-galactosidase Activity in Whole Mouse Embryos at JoVE.com

Tracing Gene Expression Through Detection of ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-galactosidase Activity in Whole Mouse Embryos

Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the method for paraffin sectioning and counterstaining. This is an easy and quick procedure to monitor gene expression during development that can also be applied to tissue sections, organs or cultured cells.

Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. ...

Research

JoVE Journal

Developmental Biology

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. ...

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its ...

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...