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>
	<li>First, prepare the electroporation mix by adding the desired plasmid DNA in ice at a final concentration of 1 &#181;g/&#181;L. Then, complete the mix by adding sucrose at 6% final concentration (dissolved in PBS), a biocompatible dye at 0.01%, and complete with PBS until a final volume of 30 &#181;L. 
	NOTE: Approximately 1-2 &#181;L of the electroporation mix is used per sample. Scale the proportions of the electroporation mix according to the number of samples in the experiment. Please remember that DNA for electroporation should be purified from bacterial culture, assuring optimal stability and minimal contaminants. For more details on the plasmids used for this study, please refer to the representative results section.</li>
	<li>Prepare Growth Medium by supplementing Dulbecco&#39;s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS), 2% Hepes buffer, and 1% penicillin/streptomycin. Keep the medium in an incubator at 37 &#176;C until use. Additionally, warm 1x PBS at 37 &#176;C, together with the growth medium.</li>
	<li>Prepare the needles by stretching a 20 &#181;m diameter glass capillary using a pipette puller with the following conditions: heat = 550; pull = 100; velocity = 250; time = 120. The final diameter after pulling should be approximately between 5-8 &#181;m.</li>
	<li>Set up the electroporator parameters to five 50 ms cycles at 40 V with intervals of 900 ms. Prepare the electrodes, mouth pipet, and blunt needle (Figure 3A). Equip the pipet with the needle to load the DNA (Figure 3A).</li>
</ol>
2. Embryo dissection and heart extraction
<ol>
	<li>Set up a cross between wild-type CD-1 mice. The next morning, select the females that exhibit a vaginal plug and note this time as embryonic day E0.5. Euthanize the animals at embryonic day E12-12.5 by cervical dislocation (following institutionally approved protocols).</li>
	<li>Carefully extract the uterus from the mouse and place it in a dry paper towel. Following this, cut the uterus using fine scissors by inserting the tip of the scissors in the mesometrial side and slowly running the edge of the blade through the whole length. Expose the deciduae and transfer them to the growth medium. For more details, please see the work by Behringer et al.20.</li>
	<li>Dissect the deciduae to extract the embryos, then place the embryos in a 60 mm Petri dish with warm growth medium. Using fine forceps, carefully cut the tail and head of the embryo so that only the trunk of the body remains. The stage of the embryos may be verified by assessing anatomical features, as described by Wong et al.21.</li>
	<li>Then, remove with caution as much tissue from the trunk of the embryo as possible without damaging the heart or the pulmonary and aorta arteries. Once the heart has been exposed, immediately place it in a new, warm growth medium and keep it in an incubator at 37 &#176;C until electroporation. 
	NOTE: Maintaining the heart&#39;s temperature is crucial for its survival. Ensure that the medium is always warm and replace it every 5-10 min. On the other hand, samples may be handled without maintaining sterility, as this will not affect the efficiency of the procedure. Please note that the growth medium contains antibiotics.</li>
</ol>
3. Heart electroporation and ex vivo culture
<ol>
	<li>Load approximately 10 &#181;L of plasmid mix (prepared in step 1.1) into the needle by carefully aspirating up with the mouth pipette.</li>
	<li>Prepare a single heart for electroporation by placing it in a clean 60 mm Petri dish with sterile 1x PBS warmed to 37 &#176;C. Place the dish under the dissecting microscope (Figure 3A).</li>
	<li>Prepare the electrodes by adjusting the distance between the positive and negative poles to approximately 4 mm (Figure 3B). 
	NOTE: 4 mm is the aperture distance in which E12.5 mouse hearts fit in the electrodes used in this work. Please consider changing this distance based on different sample sizes or electrode specifications.</li>
	<li>Gently puncture the most superficial layer of the heart with the needle in the mouth pipet. Carefully pipet 1-2 &#181;L of the plasmid mix and remove the needle. Multiple injections may be performed to ensure optimal penetration, as well as to expand the number of electroporated cells.</li>
	<li>Hold the electrodes in place so that the heart is located in between both poles (Figure 3C). Then, electroporate the heart with the conditions specified above. 
	NOTE: To ensure maximum electroporation efficiency, it is possible to switch the polarity of the electrodes and give another pulse.</li>
	<li>Transfer the electroporated heart to a new 12-well plate containing 1 mL of growth medium and place it at 37 &#176;C, 5% CO2.</li>
	<li>Repeat steps 3.1 to 3.6&#160;until all the samples in the experiment are electroporated. Finally, incubate all the hearts in an incubator at 37 &#176;C, 5% CO2, for 24&#160;h or until analysis. 
	NOTE: In our hands, hearts could be left in culture for up to 48 h without apparent signs of deterioration (Supplementary Movie 1; Supplementary Movie 2; Supplementary Figure 1).</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66754/66754fig03.jpg" /> 
Figure 3: Electroporation setup. (A) Setup used for electroporation. Red arrows indicate the electroporator, electrodes, and Petri dish where hearts are electroporated. (A&#39;) Close detail of the electroporation needle as highlighted in (A) by the white rectangle. (B) Detail of the adjusted distance of the electrodes used in E12.5 hearts. (C) Schematic representation of the electroporation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66754/66754fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Cell sorting and immunohistochemistry analysis
<ol>
	<li>For the cell sorting analysis of the samples, prepare a digestion medium to dissociate the tissue by mixing together 400 &#181;L of liberase (5 mg/ml), 300 &#181;L of dispase (0.1 g/mL), 4 &#181;L of CaCl2(0.4 &#181;M), 50 &#181;L of MgCl2(5 &#181;M) and complete until 10 mL of DMEM. Keep on ice until use. Each heart needs approximately 1 mL of Digestion Medium. Adjust the volumes according to the number of samples. 
	NOTE: For immunohistochemistry, directly fix the hearts in 1.5 mL of 4% PFA for 2 h at RT or overnight at 4 &#176;C. The next day, wash the hearts 3x with PBS and proceed to downstream histological and immunostaining procedures. For a detailed immunohistochemistry protocol, refer to Morris et al.22.</li>
	<li>Transfer each heart in culture to a new tube containing 1 mL of ice-cold digestion medium.</li>
	<li>To facilitate dissociation, chop the tissue by gently applying pleasure with a syringe 3-4 times and incubate immediately in a hot plate at 37 &#176;C, 600 rpm for 45 min.</li>
	<li>Filter the digestion mix through a 70 &#181;m cell strainer into a new tube and let it empty by gravity flow. Then, rescue the pass-through volume and filter it again using a 40 &#181;m cell strainer into a new 50 mL tube.</li>
	<li>Add 500 &#181;L of fetal bovine serum (FBS) and complete up to 30 mL using cold DMEM.</li>
	<li>Centrifuge the mix at 240 x g for 10 min at room temperature.</li>
	<li>Discard the supernatant and place the 50 mL tube upside down on a paper towel to dry completely. 
	NOTE: This is a convenient point to pause the experiment by freezing the cell pellet. If so, resuspend the cells with 10% DMSO in FBS and store at -80 &#176;C.</li>
	<li>For cell sorting, resuspend the cells in 300 &#181;L of sorting buffer (1% FBS, 1% penicillin/streptomycin in PBS) and add 0.3 &#181;L of DAPI (1000x). Perform cell sorting.</li>
</ol>

Electroporation-Based Transgenesis of Cardiac Cells in Mouse Hearts

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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 through RNA interference26. The rapid assessment of gene GOF or LOF is particularly advantageous, especially considering the large transcriptomic heterogeneity of epicardial cells12, making it impractical to generate transgenic animals to evaluate each candidate gene individually.
Because this protocol utilizes ex vivo culture, it offers the possibility to investigate the fate of epicardial cells within a temporal window. The developing epicardium plays a pivotal role in shaping distinct regions within the adult heart13,27,28; thus, understanding its contributions to heart development is instrumental in dissecting the basis of congenital cardiac malformations. While this article does not delve into this application, the guidelines outlined in the protocol could potentially be helpful for future fate-mapping studies of the epicardium
Despite the practical utility of the current method, certain limitations need to be acknowledged. Since this methodology relies on transient transgenesis, plasmid expression can be lost or diminished over time, limiting the range of the protocol to study a specific developmental window29. However, in this study, GFP overexpression was maintained throughout the entire duration of the post-electroporation cultures (48 hpe). Additionally, potential limitations in current ex vivo culture technologies hinder the method&#39;s applicability for assessing late embryonic or postnatal developmental stages, especially where ex vivo culture after electroporation becomes challenging30. Although more sophisticated methods of ex vivo embryo culture exist31,32, they may require the use of equipment that is less accessible to researchers and may not guarantee the survival of the sample.
While the protocol presented here is designed to be as simple as possible to enhance reproducibility, it includes critical steps that are instrumental for success. Firstly, embryo dissection must be conducted carefully and swiftly to minimize heart damage, which could compromise the organ&#39;s survival after electroporation. Special care during this step, along with the use of optimal dissection equipment, is strongly recommended. Another critical aspect of the protocol is performing the electroporation itself, as inadequate plasmid injection may result in undesirable outcomes. Therefore, it is advisable to perform multiple injections on a single heart, and injecting the sample in a rather superficial manner could be beneficial to enhance organ survival. Finally, cell dissociation during heart processing needs to be rigorously performed, adhering to the times provided in the protocol to maximize cell viability.
In conclusion, identifying key genes in heart formation is instrumental in understanding the underlying mechanisms of cardiac congenital diseases and regeneration. The methodology established here allows for rapid screening of gene gain-of-function (GOF) and loss-of-function (LOF) effects in the developing heart. While acknowledging certain limitations, this method exhibits considerable potential for extrapolation to different stages of development or for mapping the fate of embryonic epicardial cells. Through the visual guidelines provided in this article, we aim to offer a valuable toolbox for assessing the immense complexity and potential of the developing epicardium.

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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/files/ftp_upload/66754/66754fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>#55 Forceps</td><td>Dumont&amp;nbsp;</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&amp;nbsp;</td><td>Grandado</td><td>SKU 8798771617573</td><td></td></tr><tr><td>40 &amp;micro;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 &amp;micro;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&#039;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 &amp;times; 10 mm</td><td>BD Falcon</td><td>351008</td><td></td></tr><tr><td>Petri dishes 60 &amp;times; 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&amp;nbsp;</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.

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 referred to as pCAG::Meis1), while a constitutive GFP-expressing plasmid (pCAG::GFP) was used as a control. Hearts were then electroporated with either pCAG::GFP only or in combination with pCAG::Meis1.
After 24 h post-electroporation (hpe), the hearts were beating and appeared to be in good condition (Figure 4A; Supplementary Movie 1; Supplementary Movie 2). To assess the viability of the electroporated cells, apoptosis was analyzed through immunohistochemical staining of caspase 3. Although some positive cells were detected within the heart, neither the myocardium nor the epicardium, where the majority of the electroporated cells are located, were found to be affected by apoptosis (Supplementary Figure 1C). When the fluorescence activity was assessed, a mosaic GFP signal was observed in almost all four chambers of the heart, indicating that this protocol is able to reach the majority of heart structures (Figure 4A). To determine which cardiac cell types were electroporated, pCAG::GFP-electroporated hearts were immunostained against markers of the epicardium, WT12, and myocardium, MF2023, and then imaged using a confocal microscope (Figure 4B). Most of the GFP+ cells were preferentially located in the external region of the heart, which largely corresponded with the WT1 signal, indicating that this method is most efficient in reaching epicardial cells (Figure 4B, white arrowheads).
On the other hand, pCAG::Meis1-electroporated or control (pCAG::GFP only) hearts were dissociated, and the cells were subjected to fluorescence-activated cell sorting (FACS) to isolate GFP+ cells from the whole organ, as described in step 4 of the protocol. RNA was extracted from the sorted cells, and reverse transcription quantitative PCR (RT-qPCR) was performed to assess the expression levels of Meis1 in the transfected cells (Figure 4C, Supplementary Table 1). Total Meis1 gene expression was normalized using the expression levels of the mouse housekeeping gene Eef224, and relative gene expression was calculated using the &#916;&#916;Ct method25. A significant upregulation of Meis1 RNA levels was observed in the hearts electroporated with the pCAG::Meis1 plasmid compared to those with only the pCAG::GFP plasmid (Figure 4C). Thus, this demonstrates the potential of the technique to overexpress genes of interest and address the molecular outcome.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66754/66754fig04.jpg" /> 
Figure 4: Molecular analysis of electroporated E12.5 hearts. (A) GFP fluorescence of one CAG::GFP-electroporated heart 24 h after electroporation. The lower image shows GFP fluorescence, while the upper image shows a merge of GFP and a bright field of the same heart. (B) Immunofluorescence of electroporated E12.5 heart histological sections. MF20 marks cardiomyocytes, while WT1 marks epicardial cells. Images on the right correspond to zoom-ins of the white rectangle. White arrowheads indicate GFP+ cells located in the epicardium, roughly delimited by a dotted white line. (C) RT-qPCR analysis of hearts electroporated with pCAG::Meis1 or pCAG::GFP (control). Each dot represents a biological replicate obtained from the pooling of three hearts together. In total, qPCR analysis was performed in triplicates with three distinct biological replicates. Statistical analysis was performed using Student&#39;s t-test (*P &#60; 0.05). Scale bars: 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66754/66754fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Heart electroporation efficiency and cell viability. (A) Expression of GFP in two distinct E12.5 hearts electroporated with the control plasmid pCAG::GFP at 24 h or 48 h post-electroporation (hpe). The GFP signal image was superimposed over a bright field image of the same heart. (B) GFP+ cell quantification in 24 hpe and 48 hpe hearts electroporated with control pCAG::GFP. Dots indicate distinct samples; solid lines indicate the mean, while error bars indicate the standard deviation. No significant difference was observed between both conditions (non-parametric t-test; P &#62; 0.9). (C) Confocal image of an E12.5 heart electroporated with pCAG::GFP immunostained against cleaved caspase 3 and MF20. The dotted line represents the myocardial layer of the heart. Images on the right are zoomed in from the white rectangle. Scale bars: 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66754/Supplementary_fig1.zip" target="_blank">Please click here to download this File.</a>
Supplementary Table 1: Primer summary and sequences. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66754/Supplementary Table 1.zip" target="_blank">Please click here to download this File.</a>
Supplementary Movie 1: pCAG::GFP-electroporated heart after 24 h. Movie capturing one heart electroporated with pCAG::GFP control plasmid 24 h after the procedure. The heart continues to beat, and electroporated cells express GFP signals. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66754/Supplementary Movie 1.zip" target="_blank">Please click here to download this Movie.</a>
Supplementary Movie 2: pCAG::GFP-electroporated heart after 48 h. Movie showing an electroporated heart with control pCAG::GFP, 48 h after electroporation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66754/Supplementary_Movie_2.zip" target="_blank">Please click here to download this Movie.</a>

Author Spotlight: Cardiac Cell Transgenesis for Rapid Gene Screening

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.

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, ...

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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

561 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.

Video: Author Spotlight: Cardiac Cell Transgenesis for Rapid Gene Screening

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimal immunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations.

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

Genetics

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

Bioengineering

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Neuroscience

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the efficiency in developing gene-knockout mice by inducing small indel mutation would be sufficient enough, the efficiency of embryo genome editing for making large-size DNA knock-in (KI) is still low. Therefore, in contrast to the direct KI method in embryos, gene targeting using embryonic stem cells (ESCs) followed by embryo injection to develop chimera mice still has several advantages (e.g., high throughput targeting in vitro, multi-allele manipulation, and Cre and flox gene manipulation can be carried out in a short period). In addition, strains with difficult-to-handle embryos in vitro, such as BALB/c, can also be used for ESC targeting. This protocol describes the optimized method for large-size DNA (several kb) KI in ESCs by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.

Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method.

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the ...

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

Biology

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the host chromosome and is passed on to progeny. Thus, they are ideal vectors for creation of stable cell lines, in vivo delivery of indicators, and transduction of single cell fertilized eggs to create transgenic animals. However, mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against lentiviral gene delivery. Lentiviruses are too large to penetrate the zona and are typically delivered by microinjection of viral particles into the perivitelline cavity, the space between the zona and the embryonic cells. The requirement for highly skilled technologists and specialized equipment has minimized the use of lentiviruses for gene delivery to mouse embryos. This article describes a protocol for permeabilizing the mouse fertilized eggs by perforating the zona with a laser. Laser-perforation does not result in any damage to embryos and allows lentiviruses to gain access to embryonic cells for gene delivery. Transduced embryos can develop into blastocyst in vitro, and if implanted in pseudopregnant mice, develop into transgenic pups. The laser used in this protocol is effective and easy to use. Genes delivered by lentiviruses stably incorporate into mouse embryonic cells and are germline transmittable. This is an alternative method for creation of transgenic mice that requires no micromanipulation and microinjection of fertilized eggs.

Mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against gene delivery. This article describes a protocol for perforating the zona with a laser to transduce embryonic cells with lentiviral vectors and to create transgenic mice.

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the ...

Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk

Heart disease is the leading cause of morbidity and mortality worldwide. Due to the ease of handling&#160;and abundance of transgenic strains, rodents have become essential models for cardiovascular research. However, spontaneous lethal cardiac arrhythmias that often cause mortality in heart disease patients are rare in rodent models of heart disease. This is primarily due to the species differences in cardiac electrical properties between human&#160;and rodents and poses a challenge to the study of cardiac arrhythmias using rodents. This protocol describes an approach to enable efficient transgene expression in mouse and rat ventricular myocardium using echocardiography-guided intramuscular injections of recombinant virus (adenovirus and adeno-associated virus). This work also outlines a method to enable reliable assessment of cardiac susceptibility to arrhythmias using isolated, Langendorff-perfused mouse and rat hearts with both adrenergic and programmed electrical stimulations. These techniques are critical for studying heart rhythm disorders associated with adverse cardiac remodeling after injuries, such as myocardial infarction.

The present protocol describes methods for transgene expression in rat and mouse hearts by direct intramyocardial injection of the virus under echocardiography guidance. Methods for the assessment of the susceptibility of hearts to ventricular arrhythmias by the programmed electrical stimulation of isolated, Langendorff-perfused hearts are also explained here.

Heart disease is the leading cause of morbidity and mortality worldwide. Due to the ease of handling&#160;and abundance of transgenic strains, rodents ...

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

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs

Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk