Expansão da embrionárias e adultas células-tronco neurais por

Hi, my name is Federico K and I'm a group leader at the Center for Regenerative Therapies in Dresden, Germany. In the following movie, we would like to show you some of the technique that we have been using in our laboratory in the, in the previous years, to control the switch of mammalian neuro stem cells from proliferation to differentiation and neurogenesis. In particular, we found that when we overexpress the cycling dependent kinase for cycling D complex, we can induce the stem cell to shorter the G one, and during this time, the cells will undergo proliferative division expanding their population instead of neurogenesis.

Interestingly, when this manipulation is done, transiently and cycling CDK go back to physiological level of expression. This expanded pool of stem cell can switch to physiological neurogenesis. As a result of their expansion, the cells will generate more minerals and we found that this principle applies both during embryonic development of the mouse cerebral cortex as well as in the adult hippocampus.

So in the next video clip, Christian Lange will show you how this manipulation can be performed in developing mouse embryos by performing in electroporation. In the second part of our video, Beja will use a conceptually similar technique, but in the adult brain, in specifically in the hippocampus by performing stereotaxic viral injection. So I hope that this technique and this kind of manipulation may be useful also for your research in your laboratories and hope that you will enjoy this video.

Hi, I am Christian Langer and today I'm gonna show you the expansion of neuros stem cells in the developing mouse cortex. By neutral exploration of CDKs and cyclings. A pregnant dam gets anodized and placed on the heated operation table.

There as a flowing in vapor is applied via a breathing mask we use as a flowing because it's constant administration allows precise and flexible timing of the operation magnet. To start the operation, I carefully shaved the belly of the mouse. The shaved area is disinfected with iodine solution postoperative and a gia.

I inject 100 microliter of the tega ing solution subcutaneously. For DNA injection I used the pullout capillary filled with D supplemented plasma DNA. I cut the closed tip of the capillary using three s.

All tools were heat sterilized to open the belly of the pregnant dam. I perform a longitudinal cut at the midline of the abdominal skin using scissors. All surgical tools have been disinfected in a glass beat heat sterilizer.

From our experience, it is sufficient to keep tools in the operation platform steril to avoid bacterial infections. The peritoneum is cut analogously to the overlying abdominal skin by making a midline longitudinal incision that opens the body cavity. The cavity is moistened with the electroporation solution.

PBS supplemented with pen strap for this infection and to ease the extraction of the uterus. After performing the lipectomy, I covered abdomen with sterile surgical drape. Using a tank retractor, I open the body cavity and carefully grab the uterus between two ces to gently pull both uterine horns out of the body cavity.

For the operation, I use a pregnant mouse at Emry A 14. At this age, the embryos are clearly visible in the uterus and the operation can be performed Without the use of a microscope. It is Very important for the survival of the embryos to keep the uterus covered with the electroporation solution throughout the operation.

For the injection of the plaid DNAI grab one yolk up carefully with one hand to manipulate the embryo inside. In such a way that I can see the cephalic hemispheres, my fingers hold the sac so that the embryo inside cannot move away from the force of the needle and I inject through the uterine wall into one hemisphere until the ventricle gets outlined in blue by the colored DNA solution. After the injection, the uterus is moistened again with the electroporation solution And the electrodes Are placed.

It is essential to place the pla pole of the electrode on the uterus above the injected hemisphere and afterwards we start the electric poses using the foot switch. The electrodes are stored in PVS during the operation to wash off salts that form under contact. During electro operation, the uterus is moistened again and they continue with the next embryo.

When all embryos are electro operated, I again use the ton retractor to open the cut in the body cavity to put the embryos very carefully back inside I use faucets or if necessary the fingers to get the uterus inside. To close the wound. I start with removing the surgical drape.

I use surgical sutures to close the cut. In the peritoneal, I pull the fathom through. I turned the needle end of the fathom two times around the forceps, grabbed the other end and pull To perform The surgical knot.

It is advisable to pull the knot really tight and then the ends are cut using scissors. This technique is performed every few millimeters until the cut in the peritoneum is completely closed. The cut in the abdominal skin gets closed.

Using surgical clamps, I use forceps to create a loop in the skin, which is then clamped together. Using this clamp applicator, the operation is finished and the animal can be removed from the grieving mask to wake up and recover. Within five to 10 minutes, the animal wakes up on the operation platform and can be transferred to its home cage.

One day after the operation, the pregnant female is sacrificed and the embryos collected successfully. Electro embryos are identified under fluorescent stereo microscope. The expression of fluorescent reporters in their forebrain shown here on a dissected brain, a cross section fully the electroporated cortex reviewed the initially targeted radio ggl cells in the ventricular zone and their differentiated progeny, basal progenitors in the sub ventricular zone and neurons in the intermediate zone and cortical plate.

Thus, differentiation of the electro cells can be determined by quantification of fluorescent cells in these different zones of the cortex or by staining with specific markers for radio clear VA progenitors and neurons. Hey, my name is Ani. Today we are going to prepare some HIV antiviruses and later I will show you how to inject them into the brain of an loud mouse.

We plated five for 10 to six to nine three T cells in each 10 centimeter dish with a total of 15 dishes using demand supplemented with 10%FCS and antibiotics. Day two is the transmission. First of all, we are going to prepare the DNA solution.

We have three different plasmids. One is current for the DSPG protein, the second one for the GPU proteins, and the last one for our functional genes, so the second one and the CDK four. In this case, we prepare massive mix of the three plasmid to add to plain DN media.

Once this is done, we mix the DNA solution. We then prepare the PI solution. The PI solution is added to the DA solution.

It is mixed and incubated for about 1530 minutes at one plant. In the meanwhile, we replace the media of our plates with fresh demand containing 15%FCS in order to have a final concentration of 10%FCS. Once the transformation mix is added, we then add to ML for dish of the transformation mix.

We gently mix it and later we lift the cells in in Cupra overnight. On day three, we treat the cells with cell butyrate to reduce expression from the CMD promoter. We first add the cell butyrate to all the dishes.

We incubate them for six, eight hours at 37 degree, and then we are going to change the media. The day four is the last day of our preparation. We collect the media from all the dishes and then in order to get rid of the cell debris, we filter it.

After we collected and filter media from all the plates we transfer in the tube, it is important to carefully balance the ultracentrifuge tubes before proceeding with the ultracentrifugation and then we ultra cent get the samples At the end of the ultracentrifugation. We carefully take our samples out and really carefully we discard the, the pallet is barely visible and it looks like a shadow at the bottom of the tube. We wash the pallet with PBS and we then incubated the ice for one hour.

The there is a suspended palate transfer these smaller tubes and ultracentrifuge again, the viral palette is resus suspend in 50 microns of PBS and liquidated. The mouth has been anesthetized as already described. Using forceps, we open the mouth and we fix it on the palate part of the stereotaxic frame.

We then fix the head with the here bars and Al is administered. As already showed. We apply cream on the ice of the mouse to avoid blindness.

We fill up a capillary with oil till the half of his length, more or less, and we want the on the nanoliter injector that we will need for the injection of the viruses. The skin on the head has been shaped and carefully disinfected as already described. In order to expose the skull, I can the skin using first scalpel and then the scissor.

With the cotton bat, I gently clean the surface of the skull. The tip of the cap now has to be placed in correspondence of the prema point. To do that as much exercise as possible, we use the help of ster microscope.

We moved the arm of the stereotaxic frame on the Y and X axis until the tip of the capability has been placed at the bgma. We then we set the value that are shown on the digital display. The widget sax frame is connected.

We then move the arm of the stereotaxic frame until we do not reach the desired coordinates shown on the display. After we place the tip of the capability on the red coordinates, we mark the point using a marker pen with an electric wheel, I open a hole on this point paying attention, not the damaged brain. From now on we start to work with viruses, so we have to take special precautions to avoid any contamination.

The para are always kept in dry eyes with the keval and we t throw it. Then I take five microliters of the barrel suspension and they leave the drop on a small piece of fin that I previously put on the air bar. I move again the arm frame til the tip of the capillary is touching the droplet of the virus suspension.

Using the digital controller of the liter injecting pub will load the battery suspension in the capillary by sucking it. We then move the capillary back to the side of injections. Once the tip of the capillary is touching the PL surface, I reset the value on the Z axis On the digital display, the left of the viruses are immediately discarded.

Please remember that viruses can be decontaminated with ethanol, so everything that could potentially encountered with the virus suspension as to be clean with ethanol. Now it is time to inject, so we move the cap in the Z axis, penetrating the tissue until we reach the bite point and then we start our injection. It is important to inject the viruses at a very low injection rate.

In this case 1 55 nanoliters per minute. After pla, we wait two minutes to avoid that flow through the needle track, and then we retract the capillary at the end. It close the skin as it's been already showed.

The garbage is flashing a S two containers and then outer plate. After the surgery, the mouse can be perfused with 4%Paraform dei. The brain can be dissected and plus XI overnight in 4%paraform day.

Eventually, DU and or tamoxifen or other components can be administered before sacrifice according to different paradigms to investigate cell cycle kinetics and to stop the expression of the viral transgene. When they are flanked byside, the brain can then be processed for immuno staining using the ER markers to identify stems and progenitor cells and neurons like SOX two GFIP nesting T two double Carin or others. And this can be used to quantify the proportion of different cell population.

With that, I think you enjoy watching a video and I hope this can be useful for your research.