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

Abstract

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.

Introduction

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 formation^1,2,3. Given the crucial role of the heart, tight regulation at the cellular and molecular levels is essential for its proper formation^4,5. 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 adulthood^7,8. 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 damage^10,11. 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's heterogeneity, housing multiple cell subpopulations with distinct transcriptomic signatures^{12,13,14,15,16}. Thus, a strategy to screen potential regulators of cardiac development should accommodate the diversity of epicardial progenitor cells.

In this sense, the mouse model'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 regeneration^17,18,19. 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.

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. Please click here to view a larger version of this figure.

Figure 2: Schematic overview of the heart electroporation protocol. Please click here to view a larger version of this figure.

Protocol

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

1. First, prepare the electroporation mix by adding the desired plasmid DNA in ice at a final concentration of 1 µg/µL. 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 µL.
   NOTE: Approximately 1-2 µL 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.
2. Prepare Growth Medium by supplementing Dulbecco'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 °C until use. Additionally, warm 1x PBS at 37 °C, together with the growth medium.
3. Prepare the needles by stretching a 20 µm 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 µm.
4. 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).

2. Embryo dissection and heart extraction

1. 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).
2. 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.
3. 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.
4. 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 °C until electroporation.
   NOTE: Maintaining the heart'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.

3. Heart electroporation and ex vivo culture

1. Load approximately 10 µL of plasmid mix (prepared in step 1.1) into the needle by carefully aspirating up with the mouth pipette.
2. Prepare a single heart for electroporation by placing it in a clean 60 mm Petri dish with sterile 1x PBS warmed to 37 °C. Place the dish under the dissecting microscope (Figure 3A).
3. 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.
4. Gently puncture the most superficial layer of the heart with the needle in the mouth pipet. Carefully pipet 1-2 µL 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.
5. 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.
6. Transfer the electroporated heart to a new 12-well plate containing 1 mL of growth medium and place it at 37 °C, 5% CO₂.
7. Repeat steps 3.1 to 3.6 until all the samples in the experiment are electroporated. Finally, incubate all the hearts in an incubator at 37 °C, 5% CO_2, for 24 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).

Figure 3: Electroporation setup. (A) Setup used for electroporation. Red arrows indicate the electroporator, electrodes, and Petri dish where hearts are electroporated. (A') 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. Please click here to view a larger version of this figure.

4. Cell sorting and immunohistochemistry analysis

1. For the cell sorting analysis of the samples, prepare a digestion medium to dissociate the tissue by mixing together 400 µL of liberase (5 mg/ml), 300 µL of dispase (0.1 g/mL), 4 µL of CaCl₂(0.4 µM), 50 µL of MgCl₂(5 µM) 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 °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.
2. Transfer each heart in culture to a new tube containing 1 mL of ice-cold digestion medium.
3. 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 °C, 600 rpm for 45 min.
4. Filter the digestion mix through a 70 µm 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 µm cell strainer into a new 50 mL tube.
5. Add 500 µL of fetal bovine serum (FBS) and complete up to 30 mL using cold DMEM.
6. Centrifuge the mix at 240 x g for 10 min at room temperature.
7. 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 °C.
8. For cell sorting, resuspend the cells in 300 µL of sorting buffer (1% FBS, 1% penicillin/streptomycin in PBS) and add 0.3 µL of DAPI (1000x). Perform cell sorting.

Representative Results

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 ΔΔCt 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.

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's t-test (*P < 0.05). Scale bars: 200 µm. Please click here to view a larger version of this figure.

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 > 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 µm. Please click here to download this File.

Supplementary Table 1: Primer summary and sequences. Please click here to download this File.

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. Please click here to download this Movie.

Supplementary Movie 2: pCAG::GFP-electroporated heart after 48 h. Movie showing an electroporated heart with control pCAG::GFP, 48 h after electroporation. Please click here to download this Movie.

Discussion

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 heart^13,27,28; 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'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 exist^31,32, 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'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.

Materials

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