The overall goal of the following experiment is to induce neo eyelets in the liver of diabetic mice by helper dependent adenoviral vector mediated gene transfer. This is achieved by generating helper dependent adenoviral vectors carrying the eyelet transcription factor neuro gene in three, and the eyelet growth factor betalin next to induce periportal neo eyelid formation in the liver, the vectors are injected into diabetic mice. The treated diabetic mice are then tested for normalization of blood glucose, plasma insulin, and the presence of insulin producing beta cells in the liver.
Results from these assays demonstrate that hyperglycemia is reversed in vector treated mice as a result of neo eyelid formation in the periportal regions of the liver. The advantage of herpa dependent avir vector is a broad trophies and a high chronic capacity. And herpa dependent adenoviral vector does not contain any avir coding region.
That's why it lacks chronic toxicity. The implication of this technique extend to his therapy of diabetes because induction of new eyelets is an effective alternative to eyelet transplantation, which is limited by donor shortage, graft failure, and allo immunity. Generally, individuals new to this method will struggle because transfection process is difficult due to large size of vector classmate.
And to minimize the harbor virus contamination, the amplification and the purification process has to be handled carefully. Helper dependent adenovirus vector production involves multiple steps that must be carefully followed for optimal results. In preparation for transfection seed, 800, 001 16 cells into each of two six centimeter dishes.
Incubate at 37 degrees Celsius, 5%carbon dioxide. After two days, the cells will be 70 to 80%confluent for transfection. On the day of the transfection.
Replace the growth medium with five milliliters of fresh, medium, and incubate the cells for three hours. Then using the pro perfection arm mammalian transfection kit from Promega Transfect, the one 16 cells with 10 micrograms of DNA encoding neuro gene in three, referred to hereafter as three or a beta cell, referred to hereafter as BTC. The next day, wash the cells twice with one milliliter of growth medium each time.
Add helper virus at 500 vector particles per cell in 0.1 milliliters of PBS with calcium and magnesium. Then place the cells in the incubator every 10 minutes. Gently rock the dishes to evenly distribute the helper virus.
After 60 minutes, add 1.5 milliliters of maintenance medium and return the dishes to the incubator for 24 hours. The next day, add another one milliliter of maintenance medium. Then using a standard light microscope, examine the cells to assess cytopathic effects.
Infected cells will become rounded and detached. Continue incubating the infected cells by day two. More than 80%of cells should show cytopathic effects at this point.
Collect the crude viral lysate by pipetting up and down, and transfer it to a five milliliter tube to the crude viral lysate. Add 10%volume of 40%sucrose. Label this tube as CVL for crude viral lysate passage zero to lice.
The cells to release viral particles freeze thaw the tube containing the crude viral lysate three times by alternating placement of the tube in a minus 80 freezer and a 37 degrees Celsius water bath. After the third freeze thaw cycle, use the medium to further amplify the vector as described in the accompanying text. The vector amplification process will yield crude viral lysate from passage five or P five of the vector.
For large scale helper dependent adenovirus production begin by freeze thawing P five from the vector amplification procedure three times as before during the freeze thawing process. Transfer one 16 cells from a three liter spinner flask into 500 milliliter conical bottles and centrifuge them at 1000 times G for five minutes. After the spin, remove all but 100 milliliters of the supernatant.
Then resus suspend the cells and transfer them to a 250 milliliter spinner flask to the resuspended cells. Add all of the P five crude viral lysate. Then add helper virus at 200 viral particles per cell and incubate for one hour at 37 degrees Celsius at 60 RPM.
Following the incubation, transfer one milliliter of the cell suspension to a well of a 12. Well plate transfer the rest of the cell suspension to a new three liter spinner flask and add two liters of suspension growth.Medium. Incubate both the flask and the plate for two days in a carbon dioxide incubator at 60 RPM over the next two days, monitor the cells in the plate for cytopathic effects.
If the cells in the plate show cytopathic effects the cells in the spinner flask also have cytopathic effects. After the incubation, collect cells by centrifugation and resuspend with 15 milliliters of 100 millimolar tris. HCL in a 50 milliliter tube labeled P six.
Store this tube at negative 80 degrees Celsius until purification. To purify the vector, add one milliliter of 5%sodium deoxy coate to the thawed tube containing P six crude viral lysate. Mix gently and incubate for 30 minutes at room temperature.
Then add magnesium chloride, RNAs A and DNAs one, and incubate at 37 degrees Celsius for one hour following the incubation centrifuge the tube at 6, 000 times G for 10 minutes. Following the spin, collect the supernatant in a new 50 milliliter tube to a sterilized NVT 65 ultracentrifuge tube. Add 2.8 milliliters of a 1.25 grams per milliliter low density cesium chloride solution.
Then using a two milliliter pipette underlay, 2.8 milliliters of a 1.41 grams per milliliter high density CM chloride solution to the tube by touching the pipette to the bottom of the tube and slowly releasing the solution. Next, using a two milliliter pipette overlay five to six milliliters of supernatant. By touching the sidewall of the tube and slowly releasing the solution, the tube must be filled to the neck.
Otherwise it may collapse during high speed centrifugation. Use 100 millimolar tris HCL to fill the tube the rest of the way if necessary. Then centrifuge at 10 degrees Celsius for 30 minutes at 237, 000 times G at 10 degrees Celsius.
After the density gradient ultracentrifugation, a single opalescent vector band is visible below the opaque cel debris. Sometimes a very faint helper band can be seen below the more prominent vector band. Wipe the tube at the lower Opalescent band with 70%ethanol.
Then using a three milliliter syringe equipped with a 22 gauge needle. Puncture the wall of the tube at the base of the vector band and collect the lower opalescent band. Transfer the collected vector material into new sterilized ultra centrifuge tubes, then overlay 1.35 grams per milliliter of cesium chloride density solution to fill the tubes to the neck.
Centrifuge at 10 degrees Celsius at 237, 000 times G overnight. After the second density gradient, ultracentrifugation collect the opalescent band as before. Transfer the band into a dialysis cassette, dialyze against three liters of sterile T tris, HCL containing magnesium chloride and sucrose at four degrees Celsius overnight the next day.
To remove the helper dependent adenovirus vector from the dialysis cassette, use a three milliliters syringe to inject two milliliters of air into the cassette from the top corner. Turn the cassette around so that the syringe and needle are at the bottom corner and keeping the cassette in the upright position withdraw the solution into the syringe. Transfer the solution which now contains purified virus into labeled cryo tubes.
Aliquot 20 microliters for physical titer and 50 microliters for DNA characterization into 1.5 milliliter micro centrifuge tubes then freeze. The cryo tubes contain the purified virus at minus 80. The purified virus should then be evaluated for tighter purity and in vitro efficacy.
As described in the accompanying document, strept Odin is used to induce diabetes. In this study as described in the accompanying document, monitor strept zos and treated mice weekly for hyperglycemia. Once blood glucose is between 250 and 500 milligrams per deciliter for 48 hours, put the mice into the tail vein restrainer for a tail vein injection.
Place the tail in a 50 milliliter tube containing warm water to dilate the tail veins. Then swab the tail with a tissue soaked in 70%ethanol. Load a one milliliter syringe with a 30 and a half gauge needle with the appropriate concentration of vector check to ensure there are no bubbles in the syringe.
Then with the thumb and forefinger, grasp the tail below the injection site and insert the needle into the tail vein. If the needle is in the vein, a flash of blood may be seen in the hub of the needle. Inject the vector slowly.
If the needle is appropriately placed, there will not be resistance during injection following the injection. Remove the needle and hold the injection site with gauze to stop any bleeding before returning the mouse to the cage. After the vector treatment monitor six hour fasting glucose and body weight weekly and monitor insulin and liver enzyme levels every two weeks.
Perform a glucose tolerance test at six weeks after treatment to test whether glucose stimulated insulin secretion is restored in vivo at three and six weeks after treatment. Harvest the liver and pancreas from the mice for analysis of tissue MNA and immuno staining to assess whether eyelet neogenesis can be induced in the liver of diabetic mice via adenoviral vector mediated gene transfer. Helper dependent adenovirus or H-D-A-D-N-G-N three and BTC vectors were prepared and purified as described in this video, the vectors were administered to streptozotocin induced diabetic mice via tail vein injection.
Empty vectors and H-D-A-D-B-T-C vectors alone served as negative controls. Blood glucose was then monitored every two days by seven to 10 days following injection. Hyperglycemia was reversed in mice injected with both HDAD and GN three and H-D-A-D-B-T-C, but not in control mice.
Glucose stimulated insulin secretion was also monitored by a glucose tolerance test performed six weeks after treatment as seen here. Insulin secretion was restored in the mice injected with both HDAD and GN three and H-D-A-D-B-T-C to assess whether the insulin was synthesized in the liver. C-peptide and insulin content were assay in the liver.
C-peptide concentrations and total insulin content were measured using a commercial ELIZA kit. The presence of eptide and insulin in molar ratios confirms that the insulin being detected in the liver is indeed being synthesized in the liver. Immunohistochemistry showed positive cells in the liver of mice treated with HDAD, NGN three and H-D-A-D-B-T-C, but not in mice treated with control vector immuno staining also demonstrated there were no residual eyelets in the pancreas of the NGN three BTC treated mice.
Eyelid specific lineage transcription factor expression was also assessed by immuno staining of the liver. As shown here. NGN three was expressed in the liver of NGN three BTC treated mice, but not in the empty vector treated mice.
Likewise, BTC was expressed at a higher level in the liver of NGN three BTC treated mice as compared to that seen with empty vector treatment as seen here. P DX one immuno staining revealed nuclear localization in the NGN three BTC treated livers, but not in the empty vector treated mouse livers. NK X 6.1 was also detected only in the NGN three BTC treated mouse livers taken.
Together, these results suggest successful induction of neo eyelid formation in the mouse liver. After watching this video, you should have a good understanding of how to generate help dependent adenoviral vectors to transfer eyelid lineage determining genes into the liver for inducing eyeline. Neogenesis Once mastered, this technique can be done in three weeks if it is performed properly.
While attempting this procedure, it's important to remember that helper virus is a first generation avir that help us. Contamination should be kept to a minimum for in vivo application.
