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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V., 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=Characterization+of+a+common+progenitor+pool+of+the+epicardium+and+myocardium.">Characterization of a common progenitor pool of the epicardium and myocardium.</a> Science. 371 (6533), eabb2986 (2021).</li><li>Sendra, M., Dom&#237;nguez, J., Torres, M., Oca&#241;a, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+complexity+of+early+heart+progenitor+cells.">Dissecting the complexity of early heart progenitor cells.</a> J Cardiovasc Dev Dis. 9 (1), 5 (2021).</li><li>Ivanovitch, K., Temi&#241;o, S., Torres, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+heart+tube+development+in+mouse+reveals+alternating+phases+of+cardiac+differentiation+and+morphogenesis.">Live imaging of heart tube development in mouse reveals alternating phases of cardiac differentiation and morphogenesis.</a> eLife. 6, e30668 (2017).</li><li>Ai, D., 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=Canonical+Wnt+signaling+functions+in+second+heart+field+to+promote+right+ventricular+growth.">Canonical Wnt signaling functions in second heart field to promote right ventricular growth.</a> PNAS. 104 (22), 9319-9324 (2007).</li><li>Zimmerman, M. 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. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeobox+gene+Meis1+modulates+cardiovascular+regeneration.">Homeobox gene Meis1 modulates cardiovascular regeneration.</a> Semin Cell Dev Biol. 100, 52-61 (2020).</li><li>Stankunas, K., 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=Pbx/Meis+deficiencies+demonstrate+multigenetic+origins+of+congenital+heart+disease.">Pbx/Meis deficiencies demonstrate multigenetic origins of congenital heart disease.</a> Circ Res. 103 (7), 702-709 (2008).</li><li>Liu, Y., 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=Transcription+factor+Meis1+act+as+a+new+regulator+of+ischemic+arrhythmias+in+mice.">Transcription factor Meis1 act as a new regulator of ischemic arrhythmias in mice.</a> J Adv Res. 39, 275-289 (2022).</li><li>Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the mouse embryo: A laboratory manual. , (2014).</li><li>Wong, M. 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A., Patterson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+ex+vivo+culture+method+for+the+embryonic+mouse+heart.">A novel ex vivo culture method for the embryonic mouse heart.</a> J Vis Exp. 75, e50359 (2013).</li></ol>

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, ...

an-efficient-transgenesis-approach-for-gene-delivery-mouse-embryonic

Research

JoVE Journal

Developmental Biology

379 Aufrufe.  Severo Ochoa Center for Molecular Biology (CBMSO). Unser Ziel ist es, eine neue Methodik zu etablieren, die die Überexpression oder Herunterregulierung eines bestimmten Gens erleichtert, um die Herzentwicklung zu verstehen. Die Transgenese der Maus ist eine große Investition von Zeit und Mühe. Eine einzelne Beurteilung von Genen ist aus diesem Grund und auch wegen der hohen Komplexität des Genoms nicht realistisch.Daher besteht ein Bedarf an einer schnellen Screening-Methode. Wir haben ein Werkzeug...