The overall goal of this procedure is to construct, produce and purify canine adenovirus type two derived vectors. This is accomplished by first cloning the gene of interest into a shuttle plasmid. The second step is to obtain a recombinant genomic plasmid by homologous recombination.
Next, the recombinant defective calf, two viruses produced and amplified in a trans complementing cell line. The final step is to purify and concentrate the resulting recombinant virus for diverse applications in vivo and in vitro. Ultimately, immunofluorescence confocal microscopy is used to show examples of viral transduction in the rodent brain.
The main advantage of these techniques over existing methods such as vectors derived from human ad neurosis is that there is no preexisting immunity against calf two in human Populations. So this method can help answer key questions in the vaccination or gene therapy fields, such as assessing the role of proteins expressed in specific brain areas. So demonstrating this procedure will be may KY and Corin Bergeron.
Two postdoctoral researchers from our laboratories Prepare for this procedure one day prior to transfection by seeding DKE one cells on a six well plate grow cells at 37 degrees Celsius and 5%CO2 in DMEM with high glucose containing 7%heat, inactivated fetal calf, serum sodium, pyruvate, penicillin, and streptomycin. On the following day, the cells should be 70 to 80%confluent for transfection digest two micrograms of pcal GOI the recombinant cav two genomic plasmid with ASC one to release the non-viral sequence of the plasmid, check the resulting restriction pattern by 0.8%Agro gel electrophoresis digestion should yield an approximately 31 kilobase pair fragment containing the recombinant genome and a two kilobase pair fragment corresponding to the plasmid backbone. Mix the digested DNA with 200 microliters of jet prime buffer and vortex for 10 seconds.
Add four microliters of jet prime and mix by vortexing for 10 seconds. Spin briefly to remove droplets and incubate for 10 minutes. At room temperature, add the transfection mix dropwise onto the DKE one cells.
Gently shake the plate to evenly distribute the mix and incubate at 37 degrees Celsius. One week after transfection collect cells and culture medium in a 15 milliliter polypropylene tube. Disrupt cells by three freeze thought cycles.
Clear the lysate by centrifugation for 10 minutes at 1, 800 times G.Collect the supernatant and store at minus 20 for subsequent virus propagation to propagate the virus infect an 80 to 90%confluent monolayer of DKE one cells grown in one well of a six well plate with 0.5 milliliters of the virus containing supernatant. Incubate at 37 degrees Celsius for one hour under mild agitation After one hour. Remove the inoculum and replace it with 1.5 milliliters of complete DMEM containing 5%Heat inactivated FCS incubate cells at 37 degrees Celsius for three to four days.
Cells should be monitored daily for appearance of a cytopathic effect or CPE. If no clear CPE is visible. Harvest cells after four days of culture and repeat the viral amplification when a clear CPE affects the majority of cells.
Usually after two to four viral amplification rounds, collect cells with culture medium and freeze thaw three times as demonstrated earlier. Infect 80 to 90%confluent. DKE one cells grown in three 10 centimeter diameter tissue culture dishes using 1.5 milliliters of virus containing supernatant per dish after three to four days when CPE is complete.
Harvest cells from these 10 centimeter diameter dishes disrupt them by three freestyle cycles and clear the lysate by centrifugation for 10 minutes At 1, 800 times. G collect supernatant and store at minus 20 for large scale Cav two amplification to begin the scaling up procedure infect 80 to 90%confluent monolayers of DKE one cells grown in 40 10 centimeter diameter tissue culture dishes for each dish use 0.1 milliliters of virus containing supernatant diluted in one milliliter of complete DMEM without FCS incubate cells at 37 degrees Celsius for three to four hours under mild agitation. After three to four hours, add five milliliters of complete DMEM supplemented with 5%FCS and incubate for three days.
After three days, harvest infected cells from the 40 10 centimeter dishes in 50 milliliter polypropylene tubes, pellet cells at 1, 200 times G for 10 minutes at four degrees Celsius. Resus suspend them in 15 milliliters of DMEM medium disrupt cells by three freeze-thaw cycles. Remove cell debris by centrifuging at 1, 800 times G for 10 minutes.
Store the supernatant at minus 20 prior to purification of viral particles for purification of viral particles. Prepare a discontinuous cesium chloride gradient in a 14 milliliter UltraClear tube. First, pour two milliliters of the higher density cesium chloride solution into the bottom of the tube.
Then slowly add two milliliters of the lower density cesium chloride solution on top of the first solution. Carefully load the viral supernatant on top of the cesium chloride gradient. Fill the tube with mineral oil up to two to three millimeters from the top centrifuge in a swinging SW 40 rotor for one hour and 30 minutes at 130, 000 times g and 18 degrees Celsius after centrifugation.
Two white bands are clearly visible at the interface between the two cesium chloride solution layers. Using a 21 gauge needle and a syringe collect by side puncture the lower band containing mature cav two particles. Try as much as possible to avoid collecting the upper band that corresponds to empty viral particles.
Prepare a continuous cesium chloride gradient in a 14 milliliter transparent tube by mixing the collective viral particles with a cesium chloride solution. Fill the tube with mineral oil up to two to three millimeters from the top centrifuge in a swinging SW 40 rotor for 18 hours at 130, 000 times G and 18 degrees. After centrifugation, collect the white calf two containing band by side puncture with a syringe as demonstrated earlier.
Again, try to avoid collecting the remaining upper band. This should be easier in this continuous gradient which separates the two bands better. The total volume of collected suspension should not exceed two milliliters.
Next equilibrate a cidex G 25 PD 10 column with 30 milliliters of PBS. Load the viral suspension onto the column and discard the flow through elute with 500 microliter fractions of PBS collect fractions five to seven, which usually contain the desalted cav two particles. The virus containing fractions can easily be identified because there are scent blessed.
Finally, add 150 microliters of glycerol to the 1.5 milliliters of CAV two suspension and store in Eloqua at minus 80 degrees Celsius to titrate cav two by endpoint dilution thaw an aliquot of purified virus on ice and perform tenfold serial dilution ranging from 10 to the minus two to 10 to the minus 12 in serum free DMEM. Add 50 microliters of each viral dilution into five wells of a 96 well plate add 1.5 times 10 to the fourth DKE one cells to each well incubate the plate at 37 degrees Celsius and 5%CO2 for five days at day five. Post-infection monitor CPE appearance by microscopic observation.
Infectious titers are determined as median tissue culture infectious doses using the read and men statistical method prior to the production of viral vectors. A recombinant CAV two genome bearing an expression cassette for the transgene is constructed using standard molecular biology techniques. First, the gene of interest is cloned in a shuttle plasmid as an expression cassette with a cytomegalovirus early promoter and a polyadenylation signal flanked by two CAV two derived genomic sequences representing the upstream and downstream recombination target sequences.
In a second step, the expression cassette is inserted from the shuttle plasmid into the CAV two genome by homologous recombination. The insertion of the GOI expression cassette will lead to the deletion of the EA and part of the E one B gene in the recombinant genome. Once the recombinant genomic plasmid has been obtained, viral particles are produced using CAV two E one complementing DKE one cells.
A clear cytopathic effect due to the large amount of viral particles produced in infected cells can readily be visualized by brightfield microscopy or by transgene expression. This example is of A GFP expressing CAV two vector. After several viral amplification rounds using fresh DKE one cells, viral particles are purified by ultracentrifugation on cesium chloride gradients.
Concentrated viral particles appear as two opalescent bands within the cesium gradient. The lower band corresponds to mature recombinant cav two that should be collected. While the upper band contains empty non-infectious particles that should be avoided.
The resulting recombinant calf two vector can be used to transduce nearly all cell types in a wide variety of species, either in vitro or in vivo. Shown here is one application example in which GFP expressing CAV two was injected by stereo taxii into different areas of the mouse central nervous system. Since this protocol yields high titer and pure, pure viral stocks injection of as little as one microliter of PBS diluted GFP expressing CAV two vector in the dentate gyrus or DG leads to 40 to 50%neuronal transduction.
Moreover, due to the ability of C two to be retrograde transported in axons injection of two microliters of PBS diluted GFP expressing CAV two vector in the striatum enables the transduction of nearly 80%of nigro stri AAL neurons in the subs, nigra pars compacta After its developments. These techniques pave the way for researchers in the fields or vaccinology or neurosciences to explore novel antigens and gene function in the brain in many diverse animal models. So don't forget that working with CAF two derived vectors can be extremely hazardous and precautious, such as proper containment and waste handling measures, including personal protective equipment and work in laminar flow hoods in BSL two laboratories should always be taken while performing this procedure.
