This approach offers an efficient way to introduce other viral antigens into the HVT genome for rapid development of recombinant vaccines. Some advantage of this technique is that a GFP expression cassette is attached to the insert first for easy visualization and then removed by Cre-LoxP system. The same approach can be used to insert more viral genes at different locations of the HVT genome, or other avian herpes viruses for the development of multivalent recombinant vaccines.
Demonstrating the procedure will be Katy Moffat, an expert in cell sorting. Na Tang, Yaoyao Zhang and Guanggang Qu, scientists from my laboratory. The day before transfection prepare chick embryo fibroblasts as outlined in the text protocol.
Seed 1.3 million cells in 2.5 milliliters of medium into each well of a six well plate. Transfect the CEF cells with 0.5 micrograms each of the two Cas9 guide RNA plasmids, and one microgram of the donor plasmid using an appropriate transfection reagent according to the manufacturer's instructions. Incubate the cells at 38.5 degrees Celsius with 5%carbon dioxide for 12 hours.
12 hours post transfection dilute the HVT virus stock with M199 culture medium to a concentration of 100, 000 plaque forming units per milliliter. Add 130 microliters of the diluted virus to each well of transfected cells. Set one well of untransfected cells as a negative control and add the same quantity of virus.
Incubate the cells at 38.5 degrees Celsius with 5%carbon dioxide for three days. The day before sorting prepare two 96 well plates by seeding 20, 000 cells into each well. Three days post infection retrieve the six well plates containing the transfected and infected CEFs.
Aspirate the medium from each well and rinse the cell sheet with PBS. Add one milliliter of 0.05%trypsin-EDTA to each well and incubate the cells at 38.5 degrees Celsius with 5%carbon dioxide for approximately five minutes. Then, resuspend the cells with 50 microliters of FBS and transfer this suspension to a 1.5 milliliter microcentrifuge tube.
Centrifuge at 200 times gravity for five minutes. Resuspend the cells in one milliliter of PBS containing 1%FBS. Use a hemocytometer to count the cells and adjust the cell density to one million cells per milliliter.
Next, transfer the cells to a polystyrene sorting tube through its strainer cap. Using a cell sorter sort the single cells expressing GFP into the preseeded 96 well plates. Incubate the plates with the sorted cells at 38.5 degrees Celsius with 5%carbon dioxide for five days.
After five days use the fluorescence microscope to check the 96 well plates and mark the wells that contain a single GFP positive plaque. Trypsinize each GFP positive well with 50 microliters of trypsin-EDTA for three minutes. Then, add 50 microliters of culture medium to resuspend the cells and transfer this suspension into one well of a six well plate with CEFs.
After three days freeze down one vial of each of the first generation of recombinant viruses in freezing medium. Store these harvested viruses in liquid nitrogen. Collect 100, 000 cells from each of the first generation of viruses.
Centrifuge the cells at 2, 000 rpm for five minutes, and discard the supernatant. Store the cells at negative 20 degrees Celsius until ready to perform DNA extraction. When ready to perform DNA extraction use 50 microliters of squishing buffer to defrost and resuspend the cell pellets.
Lyse the samples at 65 degrees Celsius for 30 minutes and then at 95 degrees Celsius for two minutes to inactivate the Proteinase K.Perform a PCR reaction with three prime junction primers using one microliter of each DNA sample as outlined in the text protocol. Load two microliters of the amplification products to one well of 1%agarose gel for gel electrophoresis. To remove the GFP gene from the recombinant virus, use a transfection reagent to transfect two microliters of Cre recombinase expression plasmid into the six well plate that has been preseeded with CEF cells.
12 hours post transfection defrost one vial of recombinant virus from liquid nitrogen. Gently resuspend the virus and seed 50 microliters into each well of transfected cells, setting one well as the negative control with the same amount of virus. Incubate at 38.5 degrees Celsius for three days.
72 hours post infection prepare the cells for sorting as previously described. Sort the single non-fluorescent cells into a 96 well plate preseeded with CEFs. Next, trypsinize the chosen plaques and resuspend the corresponding cells as outlined in the text protocol.
Pass half of the cells into each well of a 96 well plate that has been preseeded with CEFs as the second generation. Centrifuge the remaining cells of each clone at 200 times gravity for five minutes. Discard the supernatant and resuspend the cells with 50 microliters of squishing buffer for DNA extraction.
Then, perform PCR with five prime junction primers using one microliter of DNA template as outlined in the text protocol. Based on the PCR results choose three to five positive clones of recombinant HVT for further passages and verification. First, infect CEFs with the second generation of recombinant HVT in a preseeded 24 well plate.
48 hours after infection remove the culture medium and add 500 microliters of 4%paraformaldehyde in PBS to fix the cells. Incubate at room temperature for 30 minutes. Next, remove the fixative and wash the cell layer three times with PBS.
Remove the PBS and add 500 microliters of 0.1%Triton X-100 to permeabilize the cells. After 15 minutes wash the cell layer three times with PBS. Add blocking buffer for one hour to block nonspecific binding.
Then, dilute the primary antibody anti-VP2 monoclonal antibody HH7 or HVT-infected chicken serum at one to 200 in blocking buffer. Add 200 microliters of the diluted primary antibody to each well and incubate at room temperature for one hour. Wash the cell layer three times with PBS.
Dilute the secondary antibody goat anti-mouse IgG Alexa 568 or goat anti-chicken IgG Alexa 488 at one to 200 in blocking buffer. Add 200 microliters of diluted secondary antibody to each well and incubate at room temperature for one hour. Then, wash the cells three times with PBS.
Use the fluorescence microscope to check the protein expression. The day before the virus expansion seed 2.6 million CEF cells into each T25 flask. Thaw at least three positive clones and add one vial of cells or viruses to each flask.
Incubate the flasks at 38.5 degrees Celsius with 5%carbon dioxide until a 50%cytopathic effect is observed. Next harvest the cells in two milliliters of culture medium. Infect 50 microliters to a new T25 flask that was preseeded with CEF cells for the next generation.
Keep passaging the recombinant virus for at least 15 generations. Using PCR analyze each generation of viruses for the presence of the VP2 sequence. Using an indirect immunofluorescence assay analyze each generation of viruses of VP2 expression.
After single cell sorting the obtained purified virus is analyzed by three prime junction PCR, which shows a PCR product of the expected size. After the excision of the GFP reporter by Cre recombinase over 50%of the plaques lost their GFP expression. The purified plaque after the GFP excision is obtained by single cell sorting and is further confirmed by five prime junction PCR which shows product of the expected size.
The protein expression is then confirmed by indirect immunofluorescence assay with VP2 specific monoclonal antibody and anti-HVT chicken serum. As expected, cells infected with the parental HVT can only be stained by anti-HVT serum. While recombinant HVT infected cells clearly show the expression of VP2 gene.
To generate a recombinant virus high transfection rate is required to maximize the chance of the virus and the insert to meet in the same cell for editing to take place. Following this procedure animal experiments can be performed to assess the immunogenicity and the efficacy of the recombinant vaccines. The development of new multivalent vector vaccines using CRISPR/Cas9 system platform described here will be highly beneficial for the poultry industry to protect against multiple poultry diseases.
