四环素调节细胞株产生高滴度的慢病毒载体，专门针对树突状细胞

The overall goal of this procedure is to generate a cell line that can produce high titers of lentiviral vectors that target dendritic cells. This is accomplished by first transducing, a gag pole rev packaging line with dendritic cell specific cbis virus glycoprotein to create A-G-P-R-S packaging line. The second step is to construct con meric DNA, encoding your gene of interest as well as a selection marker, and then transfect the GPRS packaging cells with the con meric DNA.

After selecting for transfected clones, individual clones are isolated and tested for their production capacity. The final step is to evaluate the quality of the produced lentiviral vectors in a mouse model. Ultimately, these lentiviral vectors are used to generate high antigen specific immune responses in mouse models of cancer or infectious disease.

The main advantage of this technique over existing methods like transient transfection, is that it can produce large quantities of lentiviral vectors in a reproducible and safe manner. The implications of this technique extend toward therapy of cancer and infectious diseases because the lentiviral vectors produced can generate very strong cytotoxic T-cell responses. Generally individual new to this method will struggle because performing a transfection requires precise handling and the care of the producer cell line.

In this procedure, 2 9 3 T cells are cultured in D 10 medium, 16 to 18 hours before transient transfection plate two times 10 to the 6 2 9 3 T cells in four milliliters of D 10 in a six centimeter tissue culture dish such that the cells approach 90%co fluency at the time of transfection incubate cells in a humidified 37 degrees Celsius incubator with 5%carbon dioxide. On the day of transfection of retroviral plasmids prepare a mixture consisting of 1.25 molar calcium chloride solution, sterile UE water, and the following plasmids P-R-X-S-V-G-M-U-P-G-G pole and P-V-S-V-G plasmid. P-R-X-S-V-G MU is a construct in which the dendritic cell specific SVG MU is cloned downstream of the P-type tet responsive element.

Add 500 microliters to XHBS to a five milliliter polystyrene round bottom tube. Then while bubbling the buffer vigorously with a glass past stir pipette, add the plasmid mixture dropwise into the two XHBS buffer. After adding the mixture, continue bubbling for another 30 seconds.

Next, add the whole mixture onto the 2 9 3 T cells in the six centimeter tissue culture dish and incubate at 37 degrees Celsius four hours after transfection. Carefully replace the medium in the culture dish with four milliliters of preheated D 10 and return the dish to the incubator Bader 48 hours after transfection begin the procedure for spin infection harvest S-V-G-M-U encoding retroviral particles in the supernatant by passing the supernatant through a 0.45 micron filter. The GPR packaging cell line used in this procedure is cultured in D 10 with one nanogram per milliliter of doxycycline and two nanograms per milliliter of puram mycin plate.

The GPR packaging cells in a 24 well dish at two times 10 to the fourth cells per well. Add filtered supernatant to GPR packaging cells in the dish and centrifuge cells for 90 minutes at 1050 times G and 25 degrees Celsius. After spin infection, replace medium with fresh D 10 with one nanogram per milliliter of doxycycline return cells to the incubator.

72 hours post transfection, expand the culture of transduced packaging cells, indeed 10 with doxycycline and puram mycin to confirm expression of S-V-G-M-U culture. The cells without doxycycline for 48 hours surface expression of S-V-G-M-U is subsequently measured with flow cytometry using anti antibi serum. To begin this procedure, digest 20 micrograms of plasmid TL 20 GFP with the restriction enzyme SFI one at 50 degrees Celsius TL 20 GFP is a self inactivating lentiviral transfer vector that encodes green fluorescent protein under the control of aurn stem cell virus promoter and the vector genome under the control of a tetracycline repressible promoter and digest 20 micrograms of plasmid P GK Blee with PFL M1 at 37 degrees Celsius.

PG k Blee is a bleomycin resistant cassette driven by a weak phosphoglycerate kinase. One PG k promoter subsequently purify the DNA fragments by AGROS gel electropheresis. The PGK bleak cassette is 1011 base pair and the TL 20 GFP vector is 6, 861 base pair ligate.

The TL 20 GFP vector and PGK blee cassette at a molar ratio of 25 to one using T four DNA Ligase incubate overnight at room temperature on the following day, purified DNA from the ligation mixture using a commercially available kit, ensure the amount of purified DNA is around five micrograms, 16 to 18 hours before transfection plate GPRS packaging cells in a six centimeter tissue culture dish such that the co fluency will be about 90%at the time of transfection. Using the calcium phosphate transfection method demonstrated earlier transfect five micrograms of the purified concat meric DNA into the GPRS packaging cells in the six centimeter tissue culture dish. Four hours after transfection.

Carefully remove the medium and replace with four milliliters of preheated D 10 with one nanogram per liter of doxycycline in 48 hours after transfection trypsin ice the cells and repl them in a 15 centimeter dish with 30 milliliters of D 10 with doxycycline, pur mycin and eosin culture. The cells for about two weeks to select transfected cells. After two weeks, cell colonies can be seen at the bottom of the dishes.

Label the cell colonies. Take 24 well tissue culture dishes, number the wells and to each well add two milliliters of D 10 containing zein, doxycycline, and puram Mycin aspirate the medium of the transduced cells. Add one drop of trypsin onto each of the colonies for less than one minute, and then add one or more drops of D 10 onto the same colonies with a pipette.

Pick up the colonies one by one and transfer them into separate wells of the 24 well tissue culture, plate culture, and expand all cell clones in D 10 containing zein, doxycycline and puram mycin for evaluation of viral producing ability to evaluate viral production of each cell. Clone trypsin eyes, the producer cells at approximately four times 10 to the six cells and plate in a six centimeter tissue culture dish in D 10 without doxycycline such that the co fluency exceeds 90%72 hours after removing doxycycline. Collect the medium daily for the titer assay and replace with fresh preheated D 10.

Harvest the viral supernatant by filtering the medium with a 0.45 micron filter plate. Target cells expressing the DC sign receptor bound by S-V-G-M-U in 96. Well culture dishes at one times 10 to the fourth cells per well as a negative control plate.

2 9 3 T cells in the same way, prepare twofold serial dilution of the viral supernatant and transfer the dilution to the plated cells. Usually six to eight dilution are sufficient to reach the linear range centrifuge cells and replace the medium. If bone marrow derived dendritic cells are used, they should be cultured in RPMI medium with 10%FBS and GM CSF four to six days post transduction.

The GFP expression in transduced cells is measured by flow cytometry and the lentiviral vector titer is calculated for the production of lentiviral vectors culture. The producer cell line LV MGFP in 15 centimeter tissue culture dishes, trypsin eyes, the producer cells and plate cells in 15 centimeter tissue culture dishes at greater than 90%Co fluency in fresh D 10 without doxycycline change medium with fresh preheated D 10 daily to concentrate the vectors, harvest the viral supernatant at the time of peak viral production as determined by the user and filter it using a 0.45 micron filter. Load the filtered supernatant into thick wall 32.5 milliliter ultracentrifuge tubes seal with perfil and centrifuge at 50, 000 times G four degrees Celsius for 90 minutes after centrifugation completely discard the supernatant and thoroughly resuspend the pellet in 50 microliters or an appropriate volume of PBS or HBSS depending on the application.

The stable cell line described in this method can produce large quantities of lentiviral vectors that are specifically targeted to dendritic cells. As shown here. Isolation of individual clones yielded stable cell lines of varying quality.

Among 26 clones tested eight produced lentiviral particles at a titer of greater than 10 to the six transduction units per milliliter, which is the typical benchmark for SVG pseudo typed lentiviral vectors produced by transient transfection. At the same time, several clones produced less than 10 to the fourth transduction units per milliliter. Therefore, it is important to isolate multiple clones in order to obtain a strong producer for subsequent applications.

One clone reliably produced lentiviral vectors at a titer of greater than 10 to the seventh transduction units per milliliter. To characterize this clone, the viral vector titer of culture, supernatants was measured each day for seven days after removal of doxycycline virus production peaked between 48 and 72 hours post induction. In addition, cells cultured in serum free media produced nearly as much virus as those cultured with 10%FBS.

Producer cells from this clone were cultured over three months and aliquots were tested periodically for virus production. No significant loss of virus production was observed. These results demonstrate that the high level of viral production was stable for multiple days unaffected by the presence or absence of serum in the culture medium and achievable in cells cultured for more than three months.

Subsequently, L-V-G-F-P was used to infect mouse bone marrow derived dendritic cells, and GFP expression was measured with flow cytometry as shown here. GFP expression was specific to CD 11 C positive cells, which include dendritic cells. To confirm that these viruses were not only effective in vitro mice were immunized with two times 10 to the seventh transduction units of purified and concentrated LV GFP.

Two weeks later, nearly 4%of all CD eight positive T cells were activated and expressed interferon gamma in response to the presentation of the GFP dominant peptide taken together, these results indicate that lentiviral vectors produced by stable dendritic cell targeted cell lines can selectively transduced dendritic cells in vitro and generate an immune response in vivo. While attempting this procedure, it's important to remember to treat the producer cells with atmo care in order to maximize your chances of isolating a high touch cell line. Don't forget that working with lentiviral vectors can be extremely hazardous and precautions such as wearing proper personal protective equipment and working in an approved biosafety cabinet should always be taken while performing this procedure.

Following this procedure, other emyo protein can be introduced into the paging cell line in order to generate the lenti vectors that target other cell types.