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

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.