This method can help to answer key questions in the field of Gene Therapy, Oncolysis and Genetic Vaccination. It facilitates understanding and modulating of vector hosts interactions of adenovirus gene transfer vectors, the most frequently used vector type in clinical trials up to date. The main advantage of this technique is that adenovirus capsulates can be specifically modified in a highly defined manner at selected positions and to variable extent by using simple chemistry.
The implications of this technique extends towards therapy because vector host modification cannot only be analyzed but also modulated in a patient oriented way. Though this method provides insight into specific vector host interactions, it may also be used for labeling and tracking of virus particles in living organisms, such as mice. Generally, individuals new to this method will struggle with the unique combination of Virology and Organic Cchemistry that requires practical knowledge from both fields.
Special demonstration of this technique is critical because here methods from Organic Chemistry meet methods from Cell Biology and Virology, including field-specific handling tricks. To begin this procedure prepare all buffers as outlined in the text protocol. Weigh one milliliter of each gradient buffer to check its density.
After sterilizing all the buffers, sparge the buffers containing TCEP with argon gas for about 30 seconds to prevent oxidation of the TCEP. Store the vials containing the solid powdered coupling moiety in larger tubes, each filled with a cushion of silica gel beads, to prevent the build up of condensing water when thawing the vials. Place these tubes into a desiccator.
Remove the air in the desiccator and replace it with argon gas. Then close each tube tightly and transfer it into a bigger container with silica gel beads. Once all the tubes have been transferred, close the container.
Store the container at 80 degrees Celsius until ready to use. When ready to thaw, place the container into a layer of silica beads in a desiccator. Allow the container to slowly warm to room temperature and then open the container.
When cells reach the optimal cytopathic affect, collect them by scraping the plates and transferring the supernatant to centrifuge tubes. Spin the cell suspension at 400 times G for 10 minutes. Next, re-suspend the cell pellet in four milliliters of Ad buffer containing 10 millimolar TCEP and transfer the resulting solution to a sterile 50 milliliter tube.
Freeze the solution in liquid nitrogen, and then thaw it in a water bath at 37 degrees Celsius. Repeat this freeze thaw cycle three times to rescue the vector. Centrifuge at 5000 G and four degrees Celsius for ten minutes.
While centrifuging set out two ultra centrifuge tubes to begin preparing two equal Cesium chloride step gradients. Add three milliliters of the lower phase to each of these tubes. Mark the level of the lower phase on the tube before loading the upper phase.
Then carefully stack five milliliters of the upper phase unto the lower phase, pipetting it very slowly down along the walls of the tube. When the gradients are prepared and centrifugation is complete load the supernatant from the sample equally over the two gradients. Fill the remaining space in each gradient tube with Ad-buffer containing ten millimolar TCEP making sure to fill the tubes all the way to the top.
Adjust the weight of the opposite tubes to a zero weight difference. And alter centrifuge for 176000 times G and 4 degrees Celsius for two hours. After this, use a clamp to fix one ultra centrifugation tubes to a stand.
Making sure to leave the area around the lower phase level marked accessible. Place the goose-necked lamp above the tube. Around the mark, at the border between the lower and upper phases, a distinct band should be visible.
Puncture the tube with a needle, and aspirate the vector band into a syringe to collect the vector. Transfer collected vector virons to a 15 milliliter tube and calculate the amount of coupling substrate needed as outlined in the text protocol. Then, transfer the vector solution to a tube of appropriate size.
And the calculated amount of coupling compounds stock solution. Gently mix by overhead rotation for one hour at room temperature. Next, adjust the total volume of the vector solution to six milliliters by adding Ad buffer.
Purify the vector from the unreacted coupling moiety using a second Cesium chloride binding step as outlined in the text protocol. Puncture the tube with a needle and aspirate the vector band into a syringe. Transfer the collected vectors to a 15 milliliter tube.
If the combined from both gradient tubes is less than 2.5 milliliters, adjust the volume to 2.5 milliliters by adding Ad buffer. Load the combined virons unto an equal upgraded Pd column and discard the flow through. Then add three milliliters of Ad buffer to the column and collect the flow through.
Add 333 microliters of glycerol to the collected flow through to obtain the final concentration of ten percent. Divide these into suitable aliquots. Making sure to include an aliquot of 20 microliters for tighter determination by OD 260 measurement.
Store the vector solution at 80 degrees Celsius until ready to use. Representative results of the effects on 293 cells indicate that vector production is successful. Cells should show morphology 40-48 hours after being inoculated with the virus vector.
Silver stained SDS phage is then used to assessed coupling efficiency. Vectors with Cysteins introduced into the capsomere pentone base both with the hexon modified and unmodified are run. The Hexon bands exhibit a shift indicating successful modification of over 90 percent of the monomers per capsid.
While attempting this procedure it is important to ensure a reducing environment for unmodofied vector particle. After modification, a regular atmosphere is suitable. Precise documentary calculations have to ensure quantitative modification of all vector particles.
The development of this technique helps research in the field of Gene Therapy to further explore safe and deficient strategies for vector delivery. Following this procedure other methods like coupling of ligands for targeting or fluorescent dyes for labeling can be performed in order to answer additional questions like receptor usage and bio distribution. Please make sure to obey your local GMO and Working Safety Regulations.
