The overall goal of this procedure is to perform injection of cells die, or DNA in embryonic mouse hearts. In order to investigate cell fat or to locally manipulate gene expression, this is accomplished by first dissecting out the embryos from a pregnant mouse and exposing the embryonic heart. Next DNA dye or cells are injected into the targeted region of interest in the heart.
Then after the embryo is incubated, the heart is removed and placed in culture. Finally, the fate of injected or labeled cells is determined via microscopy. Ultimately, the fate of injected, labeled, or genetically modified cells is monitored by confocal microscopy or light and epi fluorescence microscopy revealing the functional importance of specific genes and cells during cardiac development.
The main advantage of this technique of existing methods like transgenesis in mouse, is that we can locally and accurately inject lab or genetically modify cells in vivo. This allows for the investigation of this cells in the proper environment. Demonstrating this procedure will be me and Emily aar, a PhD student from a laboratory.
After preparing equipment, reagents and solutions, according to the text protocol, collect E 9.5 and E 10.5 embryos from a euthanized pregnant mouse by first using 70%ethanol to sterilize the chest, make a ventral midline incision to open the abdomen and retrieve the uterine horn before transferring it into room temperature PBS. After two additional washes in PBS, transfer it to a silicone filled Petri dish. With M 16 medium, use insect pins to pin the uterine horn to the silicone layer so that the vascularized side of the uterus is underneath.
Then use fine forceps to cut the surface of the uterus and cap the deciduous at one millimeter below the surface. Spread out the walls of the decidua and gently retrieve the embryo in the yolk sack. Store it in M 16, medium at 37 degrees Celsius prior to cell injection to carry out injections.
First, use a Hamilton syringe to fill a glass pipette from the back with DNA or cells, and shake the pipette to remove bubbles. At the tip, place the pipette on the microinjection holder and set the injector according to the following settings. Next, pin the embryo to the silicone bottom through the yolk sack without touching the embryo proper.
Then identify the region of the heart and open the sack just above this region. Add four pins around the embryo to prevent any motion during cell injection. Using the micro manipulator set to manual, slowly position the pipette tip at a 45 degree angle above the pericardium, just above the region of the heart to be injected.
Move the pipette into the region of interest, trigger the injection and take out the pipette as quickly as possible. Make sure that the heart is properly beating after the DNA or cell injection Using the injection protocol described in this video to study the epithelial to mesenchyme transition and E 9.5 A VC explan was injected with an S-H-R-N-A that downregulates a protein required for endothelial mesenchymal transition of endocardial cells. The control embryo was injected with an empty backbone vector, likewise, HUES cell derived endothelial pre valvular cells expressing GFP under the control of the SOX nine promoter were injected into the A VC at E 10.5, followed by 48 hours of explanted heart culture.
These cells acquire markers of EMT, such as periostin, similar to the endogenous endocardial cells. The ex vivo approaches demonstrated here are relatively easy, but limited to a maximum short-term follow up of 48 to 72 hours. However, the ultrasound guided injection procedure described in the text protocol provides a technically more challenging approach, but with the option of long even postnatal in utero follow-up.
The in utero injection of dye or viral vectors to label cells in specific cardiac regions is shown here. With this method, specific labeling of epicardial cells can be achieved with fluorescent dyes or virus, and the method can be expanded upon with cells or alternative injectables. Following this procedure has a method like bryo culture can be performed in order to answer additional questions like the long term effect of a gene, a molecule, or long listing readout, such as tissue morphogenesis after its development.
This technique paved the way for researcher in the field of cardiac developmental biology and genetics. It enables us to explore the world of new genes as well to investigate human cell fate in the proper embryonic context. This is very useful when a transgenic mouse line is not available.
