Een eenvoudige en efficiënte methode voor In Vivo Cardiac-specifieke genetische manipulatie door Intramyocardial injectie in muizen

The overall goal of this procedure is to achieve cardiac-specific gene manipulation in vivo, by intramyocardial adenovirus injection in adult mice. This method can help answer key questions in the field of cardiovascular research about the molecular mechanisms and align cardiac injury, and the efficacy of gene therapy. The main advantages of this technique are that it is free from ventilation, that the mice recover quickly from surgery and that it is reproducible and easy to perform.

So now, let's get started. On the day of the procedure, warm the purified adenovirus vector solution from negative 80 degrees Celsius, storage on ice. When the adenovirus has thawed, put on a face mask, a sterile gown, and sterile gloves.

And spray the adenovirus tube with 75%ethanol. Transfer the virus to a sterile laminar flow hood, and aspirate 50 microliters of the virus solution into a 50 microliter, sterilized Hamilton syringe. Attach a 30 gauge needle to the syringe.

Turn the syringe into the upright position. Gently depress the plunger, until a drop of solution is visible at the needle tip, to fill the needle tip with virus. Then, carefully aspirate another 30 microliters of adenovirus solution into the syringe, and place the syringe on ice.

To inject the adenovirus into a mouse heart, confirm the appropriate level of sedation by a lack of response to toe pinch, and apply ointment to the animal's eyes. Shave the upper chest and abdomen of the animal. Apply commercially available depilatory cream to the exposed skin.

Using wet gauze to remove the cream and any remaining hair after one minute. Using three iodine chlorhexidine based antiseptic scrubs, sterile the lower left side of the chest. Then, cover the animal with a surgical drape, with a hole cut over the surgical area, and put on a new pair of sterile gloves.

Next, using sterile technique and instruments, make a 0.5 centimeter skin incision from the xiphoid to the axilla, and use forceps and a Micro Mosquito hemostat to bluntly dissect the pectoral major and minor muscles. Retract the major and minor pectoral muscles to expose the intercostal space. And use the Micro Mosquito hemostat to pierce through and open the fourth intercostal space.

Gently press against the right side of the chest wall with the index finger of the non-dominant hand to move the heart towards the incision. Then carefully secure the externalized heart with the index finger and thumb of the same hand, and use the dominant hand to inject 10 microliters of adenovirus solution into the myocardium of each the ventral, dorsal, and lateral walls of the left ventricle. After the last injection, immediately return the heart to the intrathoracic space.

And gently press the chest wall towards the incision site, to manually evacuate any air from the intrathoracic space. Close the skin with a five ott silk suture, in a horizontal mattress pattern, and place the mouse on a 37 degree Celsius heat pad with monitoring until full recovery. Then, sterilize the used Hamilton syringe and 30 gauge needle in a high temperature, high pressure sterilizer, and discard the sterilized needle in an appropriate sharps container.

Five days after intramyocardial injection of adenovirus enclosing luciferase, in vivo imaging indicates a robust overexpression of luciferase, specifically in the heart, that can be confirmed by Western blot analysis, suggesting the absence of non-target organ transduction. Western blot analysis also indicates significantly increased vitamin D receptor expression. And the left ventricle of mice injected with adenovirus encoding vitamin D receptor.

With a reduced expression of the receptor evident in animals injected with adenovirus encoding short hair pin targeting RNA vitamin D receptor. By contrast, vitamin D receptor expression is not significantly changed in the right ventricles of either left ventricle-injected animal. Once mastered, this method can be finished within two minutes, if it is performed properly.

Following this procedure, other methods such cardiac cell injection can be performed to answer additional questions, such as the efficacy of specific cell therapies in myocardial infarctions.