Yenidoğan Fareler subventricular Bölgesi içine Plazmid DNA Transpozon Aracılı Entegrasyon Glioblastoma'nın Roman modelleri oluşturmak için

The goal of this procedure is to create novel models for glioblastomas and ultimately develop novel therapeutic approaches by inducing the formation of glioblastomas harboring specific genetic mutations in mice. This is accomplished by first creating transposon plasmids, encoding the specific genes of interest. The next steps are to prepare the plasmids with a transfection reagent and inject them into the lateral ventricle of neonatal mice.

Uptake in tumor progression are then monitored by luminescence. When the animals become more abundant, they’re sacrificed for histological and immunohistochemical analysis. Thus, new glioblastomas with specific genetic alterations are induced in wild type or targeted genetic backgrounds.

These tumors can be studied in vivo or used in vitro to generate new cell lines amenable to biochemical analysis and cytotoxicity experiments using chemotherapeutic agents. The main advantage of this technique over other methods, like for example, orthotopic injections of glioblastoma cell line, is that tumors are induced by the malignant transformation of cells endogenous to one immunocompetent animal using genetic alterations that are very specifically chosen. This process reproduces the etiology of the human disease more closely and recapitulates the salient histopathological features of human GBM.

This method can help answer key question in the field of neuro-oncology, such as what are the driver mutations responsible for inducing different types of glioblastoma multiforme, classical, pre neural, neural chy in adults or in pediatric tumors. The implications of this technique extend towards therapy of GPM since novel chemotherapeutics. Radiation immune therapies or a combination thereof can be relatively quickly tested in vivo or in vitro using this model.

I first had the idea for this method when I discussed with my colleague, Dr.John at the University of Minnesota, who had developed the initial brain tumor model using the sleeping beauty transposes mediated integration of oncogenes, an inhibitor of tumor suppressor genes into the host genomic DNA. I thought that this methodology would be very powerful to study the impact of new mutations in tumor progression and aid in developing targeted therapies for brain cancer Three to four weeks prior to the experiment, set up a mouse breeding cage with one male and one female, include a colored plastic igloo to enrich the environment and increase the chances of mating. Remove the male once a pregnancy has been confirmed, and 18 days after the mating day, start monitoring the female for delivery.

Make sure that a surrogate mother is available around the time of delivery. On the first postnatal day, perform the intraventricular injections. Prepare the injection solution by first Ali, quoting 20 microliters of the DNA mix containing the transpose on plasmids at a final concentration of 0.5 micrograms of DNA per microliter.

Next at a 20 microliter aliquot of PEI solution to the DNA solution and let them incubate at room temperature for 20 minutes to an hour after which the mix should be stored on ice. For the injection needle, attach a 10 microliter syringe to a 30 gauge hypodermic needle, beveled at 12.5 degrees. Then attach the syringe to a micro pump with automatic injection.

Test the setup with 10 microliters of water and be sure to empty the syringe. Now, cool down the neonatal stereotaxic stage with a slurry of dry ice and alcohol. When it is cooled to between two and eight degrees Celsius, proceed with the injections.

First anesthetize a pup by placing it on ice for two minutes. While the pup is on ice, load the syringe with the injection solution. Once anesthetized, transfer the pup to the frame and immobilize its head between the gauze covered ear bars.

Check that the dorsal side of the skull is horizontal and parallel to the frame surface. Also, the cranial sutures should be clearly visible. If the head of the pup is not immobilized firmly enough, it will move during the injection.

Whereas if it is squeezed too firmly between the ear bars of the frame, the lambda will not be visible and the pressure within the head will force the injected solution to leak out With the pup gripped firmly by the frame, wipe the head off with 70%ethanol, then lower the needle and adjust its position so it contacts the lambda. Next, raise the needle and move it 0.8 millimeters laterally and 1.5 millimeters tally. Then lower it again until it slightly dimples the skin.

Record the coordinates of this position. Then lower the needle, 1.5 millimeters through the skin and skull and into the cortex at the lateral ventricle there. Inject 0.75 microliters over 90 seconds, and wait a minute to let the solution disperse while waiting.

The next pup to be injected can be anesthetized. After the weight, slowly raise the needle, then transfer the pup to a recovery location under a heating lamp. The time from anesthetization to warming must be less than 10 minutes.

The quicker the better monitor its breathing, and if necessary, gently rub the limbs to stimulate breathing. Once the pup is warmed up, has a rosy color and steady breath, return it to its mother. This usually takes five to seven minutes from the injection.

If several pups are injected, they should all be recovered before returning any to the mother. If after half an hour all the pups aren’t ready, then return half of them to her and then return the rest later at the end of the procedure, monitor the dam. After returning the pups.

If she is not responsive to the pups, add the surrogate mother to the cage. 24 to 72 hours after the injection, monitor the plasmid uptake and transfer all the pups to six well tissue culture dishes one pup per well. Then load a one milliliter syringe with 30 milligrams per milliliter of Lucifer in saline or PBS and attach a 30 gauge hypodermic needle.

Inject 30 microliters of solution subcutaneously between the shoulder blades of each pup to avoid injuring the pup, inject the solution under a small fold of skin. Immediately transfer the pups to an in vivo bioluminescence imaging system. Set up a sequence to acquire four consecutive images at one minute intervals with automatic exposure, large benning and an aperture of one.Later.

After about 17 days, the animals might start forming macroscopic tumors detectable by bioluminescence and histology. It may also take up to six weeks to monitor the tumors. Inject 100 microliters of Luciferian solution intraperitoneal with a 26 gauge needle.

After three minutes, anesthetize the animals in an ISO fluorine oxygen anesthesia chamber, and place the animals in the imaging chamber fitted with cones to disperse the ISO fluorine. Acquire a series of six images at two minute intervals with an automatic exposure time, medium binning, and an open aperture F-stop of one. The region of interest is typically an oval over the head, measured the luminescence intensity in each animal using the same settings to characterize histopathological features of SB induced glioblastomas.

C 57 black six neonatal mice were injected at P one with a plasmid and coating luciferase in combination with NRA S and SV 40 large T plasmids Inod transposons with oncogenic DNA. Alternatively, a plasmid encoding a short hairpin P 53 with PDGF beta, and A GFP reporter was injected in combination with NRAS. Pseudo palis necrosis was evident as was perivascular and diffuse into the brain.

Parenchyma glioblastomas were also monitored with bioluminescence. Animals usually succumb to tumor burden. When luminescence reached an intensity of 10 to the seventh to 10 to the ninth calibrated units.

The median survival of animals was predictably dependent on the combinations of oncogenic transposons injected into the neonatal brain. The most aggressive tumors were induced with NRS and SV 40 large T antigen de novo tumors were characterized by their expression of characteristic molecules. Nascent tumors made to express GFP induced with SHP 53 and nras expressed the neural stem cell marker neston, and some also expressed glio fibrillary acidic protein.

After 56 days, a tumor was found surrounded by numerous GFAP positive astrocytes. These tumor cells expressed nein but not GFAP. After watching this video, you should have a good understanding of how to induce the formation of glioblastomas, harboring specific genetic mutations through intraventricular injections into neonatal mice, and how to monitor the progression of the disease with bioluminescence.

In the article, you will find details on how to easily clone new trans pro plasmid, encoding genes of interest, and also how to monitor the tumors and analyze them with histology and immunohistochemistry. In addition, you will find a detailed method on how to generate new glioblastoma cell lines from animals with genetically engineering tumors. We hope that this article will help speed the discovery of new therapies for this devastating human disease.