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 begin, use a pipette puller to stretch a 20-micrometer wide glass capillary. Place an extracted mouse uterus on a dry paper towel, then insert the tip of a pair of fine scissors into the mesometrial side of the uterus, and slowly run the edge of the blade through the whole length and cut it.
Now expose and cut the decidui. Transfer them into a dish with growth medium. Dissect the decidui to extract the embryos.
Transfer the embryos into a 60-millimeter Petri dish containing warm growth medium. With a pair of fine forceps, carefully cut the tail and the head of the embryo and leave only the trunk of the body. Carefully remove as much tissue as possible from the trunk without damaging the heart or pulmonary and aorta arteries.
Insert a new stretched-out needle into the tip of the mouth pipette and carefully aspirate and load 10 microliters of a prepared plasmid mix into the stretched-out needle. Place a single heart into a clean 60-millimeter Petri dish containing prewarmed PBS. Place the dish under a dissecting microscope.
Adjust the distance between the poles of positive and negative electrodes to about four millimeters. Next, use a needle in a mouth pipette to gently puncture the most superficial layer of the heart. Transfer one to two microliters of the plasmid mix into the heart;remove the needle carefully.
Hold the electrode in place so that the heart is located between both poles, then electroporate the heart. Transfer the electroporated heart to a new 12-well plate containing one milliliter of growth medium. Incubate the plate at 37 degrees Celsius under 5%carbon dioxide until analysis.
To begin, obtain electroporated mouse hearts extracted from wild type CD-1 mice 12 to 12.5 days after crossing. Transfer a heart into a tube containing one milliliter of ice-cold digestion medium. With a syringe apply gentle pressure on the heart a few times to chop it.
Incubate the chopped tissue on a hot plate at 37 degrees Celsius for 45 minutes at 600 rotations per minute, then filter the digestion mixture through a 70-micrometer cell strainer into a new tube. Next, filter the pass-through volume again with a 40-micrometer cell strainer into a new 50-milliliter tube. Pipette 500 microliters of the FBS into the filtrate.
Make up the volume up to 30 milliliters with cold DMEM. Centrifuge the suspension at 240 g for 10 minutes at room temperature. Discard the supernatant, then place the tube upside down on a paper towel to dry completely.
For cell sorting, resuspend the cells in 300 microliters of sorting buffer. Finally, add 0.3 microliters of DAPI and perform cell sorting. The hearts were beating and appeared to be in good condition.
24 hours post electroporation. The myocardium and epicardium did not show any signs of apoptosis. Analysis of the fluorescence activity showed a mosaic GFP signal in almost all four chambers of the heart.
