The overall goal of this procedure is to produce lentiviral vectors for delivering transgenes into cells from the central nervous system. This is accomplished by first cot, transecting lentiviral transfer plasmids in helper plasmids into 2 93 T cells using the calcium phosphate precipitation principle. The second step of the procedure is to purify and concentrate the vector supinate by ultracentrifugation through a 20%sucrose cushion.
The third step is to determine the titer of the vector by fax analysis or real-time quantitative PCR. The final step of the procedure is to transduce the vector into target cells. Ultimately, transduction efficiency in cell type specificity can be shown by immuno staining of the target cells expressing the transgene.
The main advantage of our technique over some other existing methods like using commercial kit in that it provides costly, effective, and a reliable approach for lentiviral vector production. So this method can provide insights into delivering transgenes into CNS cells. It can also be applied to other cell type in vivo and in vitro Lentiviral vectors are produced by cot, transfection of plasmids into 2 9 3 T cells.
The 2 9 3 T cell line should be maintained in modified DMEM for this experiment. Seed ten one hundred millimeter culture dishes with 2 9 3 T cells at 30 to 40%confluence. After 20 to 24 hours of culture, the cells should be at about 80%confluence and therefore ready for transfection.
For transfection, add all the required plasmids to a 50 milliliter tube and fill the tube to 4.4 milliliters with TE 7 9 10. Then add 0.6 milliliters of two molar calcium chloride and mix the tube gently prepare another 50 milliliter tube with five milliliters of two XHBS. Add the plasmid and calcium chloride mixture to the HBS Dropwise while vortexing the mixture.
Now over the next 30 minutes, allow a precipitate deform at room temperature. Collect the culture dishes from the incubator and after the precipitate forms suspended. Using a vortex, add one milliliter of the suspension to each dish dropwise.
While gently swirling the medium in the dish. Shake the tube briefly before transferring the suspension to the next dish. Return the loaded plates to the incubator.
Allow the plasmids to transfect the cells for five hours. After five hours, replace the media with six milliliters of media containing six millimolar. Sodium butyrate.
Then return the cultures to the incubator on the following day. If a ubiquitously expressed fluorescent reporter was used, check for its expression under a fluorescent microscope. Usually over 80%of the cells express the reporter gene.
Two days after the transfection collect the SANE from the dishes into two 50 milliliter tubes, which should amount to about 30 milliliters per tube. At this point, the supernatant can be stored at minus 80 degrees Celsius before proceeding. Start by centrifuging the supernat collected from the transfection plates to remove any cell debris.
Meanwhile, attach a 60 milliliter syringe to a 0.2 micron SFCA syringe filter. Next, decant the super named into the syringe. Then push the plug to filter the super named into a 38 milliliter polymer centrifuge tube that fits a swing rotor.
Now load a five milliliter pipette with five milliliters of 20%sucrose in PBS. Insert the pipette to the bottom of the centrifuge tube containing supernat, and slowly add the sucrose solution under the vector supernat. Now spin the SNAT for four hours in a Beckman SW 28 immediately after the spin decant and discard the supernat and resuspend the vector containing pellet in 150 microliters of 4%lactose in PBS collect all the suspensions into a single 1.5 milliliter tube and allow it to chill for 15 minutes on ice.
Next, mix the suspension by pipetting, followed by spinning it down in a micro centrifuge at full speed For one minute, transfer the super name to a fresh 1.5 milliliter tube and divide the concentrated vector into 20 microliter aliquots for storage at minus 80 degrees Celsius. To titrate the vector first seed HT 10 80 cells into a 12 well plate with one milliliter of supplemented DMEM in each well. After an overnight culture, count the cells from one well and score the cell number.
Now, make a fivefold serial dilution series of the concentrated vector into culture medium. Then add one microliter of each diluted vector to a separate well, the samples may be duplicated, but leave at least one well without vector as a negative control. Next, add one microliter of poly brain to every well and mix the plate with gentle shaking.
Return the plate to the incubator for 48 hours. Two days later, remove the media from each well and wash the cells with PPS. Then tryps anize the cells and resuspend them in one milliliter culture medium by pipetting transfer cell suspensions to 1.5 milliliter tubes and centrifuge them to collect the cell pellets.
If the vectors contain a fluorescent reporter gene resuspend the pellet in 300 microliters of 3.7%formaldehyde in PBS, then determine the percentage of positive cells by fax analysis and calculate the titer. If a fluorescent reporter gene was not used, extract the genomic DNA from the cells using a commercially available kit. Then amplify the vector sequence using real-time PCR.
Create a standard curve by amplifying the template sequence from plasmid of a known copy number in tenfold dilution series. Then calculate the titer as integration units per milliliter. Prepare neocortical cultures containing neurons and glia from mouse cortices using a previously described two step procedure in a 24 well tissue culture plate in supplemented MEM after five days.
In vitro, add 10 micromolar RSC to inhibit non neuronal cell division. Then continue the culture for two days, being careful not to let the cells dry out during media changes. After two days, replace the RC containing medium with half a milliliter of warm freshly made medium.
Then add the vector at the desired MOI to the culture and continue culturing the cells for 24 hours. After 24 hours, replace the medium and after 48 hours, if there is a reporter, gene check cells under fluorescent microscope, reporter expression should be visible in neurons two to seven days after transfection. Depending on the vector design and dose titers of lentiviral vectors produced with this protocol range from 10 to the eighth to 10 to the 10th.
Integration units per milliliter, which is suitable for in vitro or in vivo transduction of many CNS cells. Thus, mu neocortical cultures were transduced with lentiviral vectors expressing GFP controlled by a synapsin promoter or A-G-F-A-P promoter. Seven days after transduction immunohistochemistry was performed in the vector carrying the synapsin promoter.
Over 90%of neurons identified as new end positive cells expressed GFP, whereas no astrocytes identified as GFAP positive cells expressed. The reporter gene in cells transduced with the vectors containing A-G-F-A-P promoter. About 80%of astrocytes expressed GFP, but none of the new and labeled neurons expressed GFP.
These results demonstrate that lentiviral vectors are very efficient at delivering transgenes to CNS cells. Furthermore, cell specific gene expression can be achieved when appropriate promoters are used. Lindsey viral vectors have been widely used in over expressing and knocking down genes of interest in the central nervous system.
Our protocol should be useful to other investigators who want to use these methods in their laboratories.
