In Vivo Microinjection and Electroporation of Mouse Testis

The aim of this procedure is to perform microinjection of genetic constructs into the mouse testis, followed by InVivo electroporation. This allows the conduction of comprehensive gene analysis with the focus on spermatogenesis and testicular function. This is accomplished by first getting access to the testis via an abdominal incision directly above the PrepU glands.

The second step is to prepare the testis attached efferent duct for microinjection by releasing the vessel from its fatty tissue. Next, the ENT duct is punctured with a loaded microinjection pipette, and the stain genetic construct solution is administered to the testicular ferous tubules. The final step is the multilateral electroporation transfection of the testis.

Ultimately, the transfected testis is extracted after three days in order to evaluate the transfection by fluorescence microscopy or by luminometer measurements depending on the utilized reported gene constructs. This combination of microinjection intellect operation technique for mouse testis as a transient transfection methods will certainly provide many advantages over existing approaches like transgenic animals or knockout mice. It ensures a fast performance, a low cost, and the need of just moderate technical skills.

This methodological approach could hopefully wise to available technique in the field of male fertility research. It might be essentially helpful for addressing a broad spectrum of issues concerning experimental genis and fertility processes. This would be particularly possible when using this method for genetic analysis in the form of gain of all loss of function experiments.

Presumably, it will be very useful also for investigating regulatory elements of, for instance, spermatogenesis specific genes. To begin the procedure, anesthetize a six to eight week old male mouse with a one-to-one mix of 10%ketamine and 2%xylazine subcutaneously. Then make sure that the mouse is under deep anesthesia by toe pinching.

Apply ointment to its eyes to prevent dryness. Next, remove the abdominal hair with an electric shaver and disinfect the surgical area afterwards. Make a ventral incision directly above the PrepU glands in the center of the abdominal area.

For that, pull the skin and conduct a small transverse cutaneous cut. Then continue along the abdominal muscular layer. Pull up the abdominal fat pads carefully to expose the attached testis and place it on a sterile paper on the abdomen.

Subsequently, use a stereo microscope to find the ENT duct for microinjection. It is the fine vessel junction between the testis and the epididymus, and is located adjacent to the testicular artery. Remove the fatty tissue that covers the ENT duct with fine forceps.

After that, place the release ductus on a sterile paper strip, ensuring the ductus is easily visible without impairing surroundings. Further on, connect the microinjection pipette, which has been loaded with the colored plasmid solution before to the micro manipulator injector unit. After that, place the micro injection needle parallel to the EENT duct with the tip pointing towards the REIT testis.

Then use fine tweezers to strip the duct over the microinjection pipette. Make sure that the pipette is kept in parallel to the duct while pulling it over. Direct the needle carefully towards the testis and stop just as it penetrates the REIT testis directly beneath the tunica albuginea.

Next, set the parameters of the micro injector. Subsequently, begin injecting the gene construct solution. Monitor the entire injection process by observing the testis filling status.

With the help of the colored plasmid solution, build the testis only up to two thirds of its volume. To enable effective electroporation. Soak the tweezer electrodes in one times PBS to ensure adequate conductance.

Then squeeze the test is slightly between the wet electrodes and measure the electrical resistance with the electro parata. Next, set the electro adequately ensuring that current can be applied to the testis with a total number of eight square pulses. At four different testis sites, perform the electroporation comprehensively around the entire testis.

After the electroporation is finished, place the testis back into the abdomen as close to its original location as possible. So the inner muscular layer was absorbable surgical suture. Then close the skin with suture clips.

Allow the mouse to recover from anesthesia. In a sterile box warmed on a 37 degrees Celsius heat pad, prepared with some paper towels. The pad should ensure the warming of only one half of the cage creating a heat gradient.

Additionally, cover the mouse with another sheet of paper towel to further reduce stress. After the animal fully wakes up, transfer it to a new clean, completely equipped cage. But further recovery, monitor the recovery process until you finally return the mouse to the animal facility.

This figure shows the whole Mount testes three days after in vivo electroporation by fluorescence detection, the transfected testes of a C 57 black six mouse show enhanced green fluorescent protein or red fluorescent protein shown. Here are the histological sections of a testis. Three days after unilateral electroporation with PE eeg, FPC one, the transfected Seminiferous tubial cells are illustrated by green fluorescence, along with their typical spherically organized structure.

Their nuclei were counterstain with two pro three in blue. While you are performing this procedure, it is important to keep in mind the processes of identifying the ENT duct and its release of its fatty tissue are quite sophisticated. Therefore, you may practice on dead animal first before accomplishing real in vivo experience.

Following the anti proceeding of microinjection electroporation and final test harvesting, many techniques are possible to be performed like Q-R-T-P-C-R ship and Western blotting. Hence, xining of tester gene are protein expression patterns as well as post translational modifications is for granted. So by the means of these tools, a vast variety of topics might be attacked like spermatogenesis, for example, with its underlying complex mechanisms and regulations.