This study was supported by grant RTI2018-097617-J-I00 from the Spanish Ministerio de Ciencia e Innovacio&#769;n and Acci&#243;n 9 from Universidad de Ja&#233;n to O.H.O. Grant PGC2018-096486-B-I00 from the Spanish Ministerio de Ciencia e Innovacio&#769;n and grant H2020-MSCA-ITN-2016-722427 from the EU Horizon 2020 program to M.T. JMG was supported by a PhD fellowship from the Spanish Ministry of Science and the Fundaci&#243;n Severo Ochoa (PRE2022-101884). Both the CNIC and CBMSO are supported by the Spanish Ministry of Science, and the CNIC is supported by the ProCNIC Foundation.

All animal procedures were approved by the CNIC Animal Experimentation Ethics Committee and conformed to current legislation, including EU Directive 2010/63EU and Recommendation 2007/526/EC, as enforced by Spanish Law under Real Decreto 53/2013. For this protocol, female wild-type CD-1 mice aged 15-21 weeks were employed. Details regarding the animals, reagents, and equipment used are listed in the Table of Materials.
1. Plasmid and tool preparation
<ol>
	<l...

Electroporation-Based Transgenesis of Cardiac Cells in Mouse Hearts

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global,+regional,+and+national+burden+of+congenital+heart+disease,+1990-2017:+A+systematic+analysis+for+the+Global+Burden+of+Disease+Study+2017.">Global, regional, and national burden of congenital heart disease, 1990-2017: A systematic analysis for the Global Burden of Disease Study 2017.</a> Lancet Chil Adolesc Heal. 4 (3), 185-200 (2020).</li><li>Xin, M., Olson, E. N., Bassel-Duby, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mending+broken+hearts:+Cardiac+development+as+a+basis+for+adult+heart+regeneration+and+repair.">Mending broken hearts: Cardiac development as a basis for adult heart regeneration and repair.</a> Nat Rev Mol Cell Biol. 14 (8), 529-541 (2013).</li><li>Porrello, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+regenerative+potential+of+the+neonatal+mouse+heart.">Transient regenerative potential of the neonatal mouse heart.</a> Science. 331 (6020), 1078-1080 (2011).</li><li>Cao, J., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epicardium+as+a+hub+for+heart+regeneration.">The epicardium as a hub for heart regeneration.</a> Nat Rev Cardiol. 15 (10), 631-647 (2018).</li><li>Zhou, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+mouse+epicardium+modulates+myocardial+injury+by+secreting+paracrine+factors.">Adult mouse epicardium modulates myocardial injury by secreting paracrine factors.</a> JCI. 121 (5), 1894-1904 (2011).</li><li>Van Wijk, B., Gunst, Q. D., Moorman, A. F. M., Van Den Hoff, M. J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+regeneration+from+activated+epicardium.">Cardiac regeneration from activated epicardium.</a> PLOS One. 7 (9), e44692 (2012).</li><li>Hesse, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+transcriptomics+defines+heterogeneity+of+epicardial+cells+and+fibroblasts+within+the+infarcted+murine+heart.">Single-cell transcriptomics defines heterogeneity of epicardial cells and fibroblasts within the infarcted murine heart.</a> eLife. 10, e65921 (2021).</li><li>Streef, T. J., Smits, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicardial+contribution+to+the+developing+and+injured+heart:+Exploring+the+Cellular+composition+of+the+epicardium.">Epicardial contribution to the developing and injured heart: Exploring the Cellular composition of the epicardium.</a> Front Cardiovasc Med. 8, 750243 (2021).</li><li>Sanchez-Fernandez, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+epicardial+cell+heterogeneity+during+cardiogenesis+and+heart+regeneration.">Understanding epicardial cell heterogeneity during cardiogenesis and heart regeneration.</a> J Cardiovasc Dev Dis. 10 (9), 376 (2023).</li><li>Quijada, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+of+endothelial+cell+positioning+and+fate+specification+by+the+epicardium.">Coordination of endothelial cell positioning and fate specification by the epicardium.</a> Nat Commun. 12 (1), 4155 (2021).</li><li>Mantri, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+single-cell+RNA+sequencing+of+developing+chicken+hearts+identifies+interplay+between+cellular+differentiation+and+morphogenesis.">Spatiotemporal single-cell RNA sequencing of developing chicken hearts identifies interplay between cellular differentiation and morphogenesis.</a> Nat Commun. 12 (1), 1771 (2021).</li><li>Paul, S., Zhang, X., He, J. 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The authors have no conflicts of interest to disclose.

Overall, the methodology described here offers a robust framework for expressing transgenic constructs in the developing epicardium (Figure 4B), as demonstrated by Meis1 overexpression (Figure 4C). With the appropriate constructs, this protocol can be used to transiently assess the impact of either gain-of-function (GOF) or loss-of-function (LOF) of a specific gene. LOF can be implemented into the technique by transfecting a plasmid targeting a candidate gene th...

The heart is the first organ formed during embryonic development. This process involves the spatiotemporal coordination of various populations of progenitor cells from distinct areas of the embryo. All this occurs while the developing heart continues to beat and function, emphasizing the remarkable coordination required for its formation1,2,3. Given the crucial role of the heart, tight regulation at the cellular and molecular levels is essential for its proper formation4,5. Identifying the mechanisms that control heart development has been of great interest, as they are crucial for unraveling congenital heart disorders, which impact a substantial number of patients worldwide6. Furthermore, comprehending heart development is pivotal in deciphering cardiac regeneration, as postnatal mammalian hearts retain a regenerative capacity that is lost or hindered in adulthood7,8. Consequently, dissecting molecular regulators of heart development is imperative to advance research efforts on congenital heart disease and cardiac regeneration.
In pursuit of this objective, there has been a growing focus on investigating the role of the epicardium in cardiac development and regeneration9. The epicardium is a thin layer of mesothelial tissue that comprises the outermost layer of the mammalian heart (Figure 1). Recent studies have shown the importance of the epicardium during cardiac injury, revealing that this tissue is able to send proliferation signals to cardiomyocytes in the affected area to mitigate the damage10,11. Despite the importance of the epicardium, conducting further molecular investigations has been challenged by its immense heterogeneity. Single-cell RNAseq experiments have revealed the epicardium&#39;s heterogeneity, housing multiple cell subpopulations with distinct transcriptomic signatures12,13,14,15,16. Thus, a strategy to screen potential regulators of cardiac development should accommodate the diversity of epicardial progenitor cells.
In this sense, the mouse model&#39;s amenability to genetic modification has facilitated the identification of numerous genes crucial for heart development, allowing the generation of mutant lines with gain-of-function (GOF) or loss-of-function (LOF) of specific genes. However, these approaches imply a considerable investment of time and experimental resources; therefore, they are impractical when assessing the roles of a large number of candidate genes. Besides, developmental genes often exert pleiotropic functions in different tissues or are required for early embryonic development, hampering the interpretation of their contribution to development in a specific process. While it is possible to target gene function at specific structures or developmental time points, this usually requires the use of more complex genetic constructions, which can be difficult to generate or are generally unavailable.
To overcome these limitations, we present a methodology to electroporate mouse embryonic hearts for transient transgenesis (Figure 2). Paired with ex vivo culture and fluorescence-activated cell sorting (FACS), this strategy demonstrates its capabilities through transient GOF of Meis1, a well-characterized gene implicated in heart development and regeneration17,18,19. In this article, other potential applications of this methodology are also explored, and its advantages and limitations are discussed, as well as compared to existing protocols for transiently modulating gene expression. We believe the framework and visual examples presented will enhance the understanding of epicardium biology during development and disease.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66754/66754fig01v2.jpg" /> 
Figure 1: Mouse embryonic heart layers. Schematic diagram of a coronal view of an E13-14 mouse embryonic heart. The three main cellular layers of the heart are represented in yellow (endocardium), red (myocardium), and blue (epicardium). The pericardium is represented in a brown line. The four chambers of the heart are abbreviated as LV, left ventricle; RV, right ventricle; LA, left atrium; RA, right atrium. <a href="https://www.jove.com/files/ftp_upload/66754/66754fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66754/66754fig02.jpg" /> 
Figure 2: Schematic overview of the heart electroporation protocol. <a href="https://www.jove.com/files/ftp_upload/66754/66754fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#55 Forceps</td>
			<td>Dumont&#160;</td>
			<td>11295-51</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well Clear Flat Bottom Multiwell Cell Culture Plate</td>
			<td>BD Falcon</td>
			<td>353043</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm vise table&#160;</td>
			<td>Grandado</td>
			<td>SKU 8798771617573</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m Cell Strainer</td>
			<td>Fischer Scientific</td>
			<td>08-771-1</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tubes</td>
			<td>BD Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m Cell Strainer</td>
			<td>Corning</td>
			<td>CLS431751</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP Policlonal Antibody</td>
			<td>Invitrogen</td>
			<td>A10262</td>
			<td>1:1000 dilution used</td>
		</tr>
		<tr>
			<td>Anti-Myosin 4 (MF20) Monoclonal Antibody</td>
			<td>Invitrogen</td>
			<td>14-6503-82</td>
			<td>1:500 dilution used</td>
		</tr>
		<tr>
			<td>CD1 Wild Type mice</td>
			<td></td>
			<td></td>
			<td>Provided by Animalary Unit (CNIC)</td>
		</tr>
		<tr>
			<td>Cleaved Caspase-3 (Asp175) Antibody</td>
			<td>Cell Signalling Technologies</td>
			<td>9661</td>
			<td>1:400 dilution used</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Cell Signalling Technologies</td>
			<td>4083</td>
			<td>1:1000 dilution used</td>
		</tr>
		<tr>
			<td>Dispase/collagenase</td>
			<td>Roche</td>
			<td>10269638001</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM - Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco</td>
			<td>10313021</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Invitrogen</td>
			<td>10438-026</td>
			<td></td>
		</tr>
		<tr>
			<td>Heracell 150i CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>51032720</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Stereoscopic Microscope S8AP0</td>
			<td>Leica</td>
			<td>11524102</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase</td>
			<td>Roche</td>
			<td>5401119001</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller Model P-97</td>
			<td>Sutter Instrument</td>
			<td>SU-P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>pCAG expression plasmid</td>
			<td>Addgene</td>
			<td>#89689</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Invitrogen</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes 35 &#215; 10 mm</td>
			<td>BD Falcon</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes 60 &#215; 15 mm</td>
			<td>BD Falcon</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>Merck</td>
			<td>P3532</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td></td>
			<td></td>
			<td>Reused from old laboratory equipment</td>
		</tr>
		<tr>
			<td>Rat Serum culture embryo, male rats SPRAGUE DAWLEY RjHan SD</td>
			<td>Janvier Labs</td>
			<td>9979</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant anti-Wilms Tumor Protein 1 (WT1) Antibody</td>
			<td>Abcam</td>
			<td>ab89901</td>
			<td>1:300 dilution used</td>
		</tr>
		<tr>
			<td>Square Wave Electroporator CUY21SC</td>
			<td>Nepa Gene</td>
			<td>CUY664-10X15</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile PBS</td>
			<td></td>
			<td></td>
			<td>Provided and autoclaved by technical unit</td>
		</tr>
		<tr>
			<td>Sucrose&#160;</td>
			<td>Millipore</td>
			<td>84100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer electrodes with variable gap</td>
			<td>Nepa Gene</td>
			<td>CUY650P5</td>
			<td></td>
		</tr>
	</tbody>
</table>

an efficient transgenesis approach for gene delivery in the mouse embryonic heart

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, and fate of these progenitors are vital for the proper development of this organ. The molecular mechanisms that govern the morphogenesis of the heart are essential for understanding the pathogenesis of congenital heart diseases and embryonic cardiac regeneration. Classical approaches to investigate these mechanisms employed the generation of transgenic mice to assess the function of specific genes during cardiac development. However, mouse transgenesis is a complex, time-consuming process that often cannot be performed to assess the role of specific genes during heart development. To address this, we have developed a protocol for efficient electroporation and culture of mouse embryonic hearts, enabling transient transgenesis to rapidly assess the effect of gain- or loss-of-function of genes involved in cardiac development. Using this methodology, we successfully overexpressed Meis1 in the embryonic heart, with a preference for epicardial cell transfection, demonstrating the capabilities of the technique.

Author Spotlight: Cardiac Cell Transgenesis for Rapid Gene Screening

An Efficient Transgenesis Approach for Gene Delivery in the Mouse Embryonic Heart

We aim to establish a new methodology that facilitates the overexpression or downregulation of a certain gene to understand heart development. Mouse transgenesis is a great investment of time and effort. Assessing genes one by one is not realistic because of this and also because of the high complexity of the genome.Hence, there is a need for a rapid method of screening. We have presented a tool available to any researcher to analyze the gain or loss of function of a candidate gene during heart development. The main advantage of our protocol is that it's much quicker and less expensive than mouse transgenesis.Perhaps it could be used to assess different genes of interest before commencing a transgenesis experiment.

To demonstrate the effectiveness of this technique in performing gain-of-function (GOF) experiments for relevant heart developmental regulators, a construct was electroporated overexpressing the Meis1 transcription factor. To achieve this, RNA was extracted from E9.5 embryos, and reverse transcription was performed to obtain complementary DNA (cDNA). Using the cDNA as a template, the Meis1 coding sequence was cloned (Supplementary Table 1) into a pCAG expression plasmid (hereafter referr...

This protocol presents a detailed methodological framework for electroporation-based transgenesis of cardiac cells in developing mouse hearts. The video assets provided here will facilitate learning of this versatile technique.

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, ...

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Research

JoVE Journal

Developmental Biology

Extraction and Electroporation of Mouse Embryo Heart as a Transgenesis Approach

To begin, use a pipette puller to stretch a 20 micrometer wide glass capillary. Place an extracted mouse uterus on a dry paper towel. Then, insert the tip of a pair of fine scissors into the myometrial side of the uterus, and slowly run the edge of the blade through the whole length and cut it.Now, expose and cut the decidui. Transfer them into a dish with growth medium. Dissect the decidui to extract the embryos.Transfer the embryos into a 60 millimeter Petri dish containing warm growth medium. With a pair of fine forceps, carefully cut the tail and the head of the embryo, and leave only the trunk of the body. Carefully remove as much tissue as possible from the trunk without damaging the heart or pulmonary and aorta arteries.Insert a new stretched out needle into the tip of the mouth pipette, and carefully aspirate and load 10 microliters of a prepared plasmid mix into the stretched out needle. Place a single heart into a clean 60 millimeter Petri dish containing prewarmed PBS. Place the dish under a dissecting microscope.Adjust the distance between the poles of positive and negative electrodes to about four millimeters. Next, use a needle in the mouth pipette to gently puncture the most superficial layer of the heart. Transfer one to two microliters of the plasmid mix into the heart.Remove the needle carefully. Hold the electrode in place so that the heart is located between both poles. Then, electroporate the heart.Transfer the electroporated heart to a new 12 well plate, containing one milliliter of growth medium. Incubate the plate at 37 degrees Celsius under 5%carbon dioxide until analysis.

Electroporated Mouse Cardiac Cell Isolation for Cell Sorting and Analysis

To begin, obtain electro operated mouse hearts extracted from wild type CD1 mice, 12 to 12.5 days after crossing. Transfer a heart into a tube containing one milliliter of ice cold digestion medium. With a syringe, apply gentle pressure on the heart a few times to chop it.Incubate the chopped tissue on a hot plate at 37 degrees Celsius for 45 minutes at 600 rotations per minute. Then filter the digestion mixture through a 70 micrometer cell strainer into a new tube. Next, filter the pass through volume again with a 40 micrometer cell strainer into a new 50 milliliter tube.Pipette 500 microliters of the FBS into the filtrate. Make up the volume up to 30 milliliters with cold DMEM. Centrifuge the suspension at 240G for 10 minutes at room temperature.Discard the supernatant. Then place the tube upside down on a paper towel to dry completely. For cell sorting, resuspend the cells in 300 microliters of sorting buffer.Finally, add 0.3 microliters of DAPI and perform cell sorting. The hearts were beating and appeared to be in good condition 24 hours post electroporation. The myocardium and epicardium did not show any signs of apoptosis.Analysis of the fluorescence activity showed a mosaic GFP signal in almost all four chambers of the heart.

380 Views.  Severo Ochoa Center for Molecular Biology (CBMSO).  This protocol presents a detailed methodological framework for electroporation-based transgenesis of cardiac cells in developing mouse hearts. The video assets provided here will facilitate learning of this versatile technique.

Electroporation-Based Transgenesis of Cardiac Cells in Mouse Hearts

Summary