The overall goal of the following experiment is to introduce oncogenes or other potentially hazardous genes into human cells using echo tropic lentivirus. This is achieved by producing pantropic lentivirus, encoding the mouse retrovirus receptor, as well as echo tropic lentivirus encoding the gene of interest cells are transduced with the mouse retrovirus receptor, which makes them susceptible to transduction with echo tropic virus. Next, the cells are transduced with echo tropic virus in order to introduce the oncogene using polymer complexation with poly brain and chondroitin sulfate to enhance transduction results are obtained that show selective transduction of human cells with tropic lentivirus based on fluorescent microscopy and facts.
The main advantage of this technique over existing methods such as gamma retroviral transduction, is that lentiviral particles can more effectively transduce many cell types, including non dividing cells such as stem cells. This method can help answer key questions in the field of cancer and stem cell research, such as understanding the function of potential oncogenes. Before beginning this procedure, consult your institutional safety official and follow their recommended safety guidelines.
Begin with a culture of healthy rapidly growing 2 9 3 T cells working in a tissue culture hood. Under aseptic conditions, resus suspend five times 10 of the 6 2 9 3 T cells in 10 milliliters of antibiotic free 2 9 3 T medium. Pipette the cell suspension into a 10 centimeter tissue culture plate and incubate the plate overnight in a tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide.
The next day, begin transfection in the later afternoon. First, collect an aliquot of Optum and the transfer packaging and envelope plasmids and allow these to warm to room temperature pipette 375 microliters of optimum into a micro centrifuge tube, and then add 25 microliters of FU gene HD transfection reagent. Ensure that the FU gene reagent is pipetted directly into the media and does not touch the side of the tube.
Next, in a second micro centrifuge tube, combine five micrograms of the transfer plasmid, 3.75 micrograms of the packaging plasmid, and 1.25 micrograms of the envelope plasmid in the required amount of optimum to bring the volume to 100 microliters. Then add the Optum Fuji mixed to the tube containing the plasmid mixture and incubate room temperature for 20 to 30 minutes. During the last few minutes of the incubation, very gently aspirate the media from the plate containing the 2 9 3 T cells and replace it with 10 milliliters of fresh antibiotic free 2 9 3 T medium.
Finally, add the FU gene plasmid mix dropwise into the media of the 10 centimeter plate. Place the plate in a tissue culture incubator set to 37 degrees Celsius and 5%carbon dioxide and incubate overnight. The next day, very gently aspirate the media from the plate and replace with fresh antibiotic-free 2 9 3 T medium.
Then replace the plate in the tissue culture incubator and incubate for a further two days, three days after the transfection aspirate the media containing the virus from the plate and filter it using a 0.45 micron low protein binding filter. After filtering tighter the virus to tighter virus, make serial dilutions of a fluorescent control virus in fresh antibiotic-free media with six micrograms per milliliter poly brain and use these dilution to transduce cells of the appropriate cell line. In a 24 well plate the next day, change the media to fresh, antibiotic-free media and then continue the incubation for a further two days to allow the cells to express the fluorescent protein.
Use facts to determine the fraction of transduced cells for each viral dilution. Then calculate the titer in transforming units per milliliter based on dilutions that yield less than 15%transduction to minimize multiple transduction events. The tits of other non fluorescent lentivirus produced in parallel are presumed to be similar to that of the fluorescent virus.
To transduce target cells Begin by selecting a multiplicative infection greater than two to ensure that most cells will be transduced. First, dilute the viral supernatant to the required multiplicity of infection in fresh complete culture medium. Supplemented with six micrograms per milliliter of poly brain to a final volume of one milliliter.
Aspirate the media from a 35 millimeter dish of target cells and add one milliliter of diluted viral supinate. Incubating the tissue culture incubator for 24 to 48 hours to allow sufficient expression of the SLC seven A one receptor if desired. Use antibiotic selection with blaster sein to select for stably transduced cells expressing SLC seven A one according to the instructions in the written procedure.
To begin toxicity determination, prepare a stock solution of 20 milligrams per milliliter chondroitin sulfate and a stock solution of 20 milligrams per milliliter. Poly brain in water and sterile filter. Excess stock solutions may be stored at minus 20 degrees Celsius until needed.
Next, determine the number of 96 well cell cultures needed to accurately determine polymer complex toxicity. Using the MTT assay, at least six concentrations of chondroitin sulfate and poly green should be tested in triplicate. In addition, unreduced cells and cells grown in serum free.
Medium should be included to provide a baseline for healthy cells and to control for growth arrest respectively. Once the number of required wells has been ascertained, plate 5, 000 target cells in each well of a 96 Well plate in parallel plate cells in a 24 well plate for microscopic or facts-based analysis of transduction efficiency in each experimental condition, allow both sets of cells to grow overnight. The next day tho an aliquot of the chondroitin sulfate stock solution and an aliquot of the poly brain stock solution.
Then dilute virus encoding GFP in antibiotic-free media containing six micrograms per milliliter poly brain to a multiplicity of infection sufficient to transduce around 10 to 50%of target cells per well. Ensure that there is enough diluted virus for each test condition plus control wells on each plate. Label a set of tubes with each chondroitin sulfate and poly green polymer concentration to be tested.
Then aliquot enough of the viral suspension for each condition in triplicate into the tubes. Next, pipette the required volume of chondroitin sulfate and poly brain stock solution to make up the test concentrations into each of the tubes in succession. Immediately after the addition of chondroitin sulfate and poly green flick the tube to mix.
Allow the virus chondroitin sulfate poly green mixtures to incubate at room temperature for five minutes. The mixture may become cloudy as viral precipitates form. Following the incubation, aspirate the media from the wells of the 96 well plate.
And then add the virus chondroitin sulfate poly green mixture to the wells as planned. Also pipette control and serum free media into the appropriate wells. Repeat the process for the 24 well plate incubate overnight in the tissue culture incubator.
Next, aspirate the virus containing media from the plates and replace with fresh medium. Return the plates to the incubator for a 48 hours After 48 hours. Remove the 24 well plate and use facts to analyze the transduction efficiency of each chondroitin sulfate and poly brain concentration.
The following day. Aspirate the media from all wells of the 96 well plate and replace with 100 microliters of MTT solution culture for three hours in a humidified incubator to allow MTT to be reduced in the mitochondria of metabolically active cells. After three hours, aspirate the MTT solution and replace with 200 microliters of solvent solution.
Incubate the plate for two hours at room temperature or until all of the purple MTT forms and precipitate is dissolved. Read the absorbence on a microplate reader at 570 nanometers, subtracting the background reading as 690 nanometers. The optimal polymer concentration will produce maximum enhancement of transduction as determined by facts with minimal effect on metabolic activity as determined by MTT for hdfs.
100 micrograms per milliliter of chondroitin sulfate. Poly brain is the optimal polymer concentration as determined by this method. First, prepare enough 100 microgram per milliliter chondroitin sulfate poly green viral sup natant mixture to transduce the required number of 35 millimeter dishes of SLC seven A one receptor expressing HDF cells Incubate the mixture for five minutes to allow polymer complexes to form.
Then aspirate the media from the receptor expressing target cells and replace with the viral mix incubate overnight at 37 degrees Celsius and 5%carbon dioxide after removing the viral mix. Wash the surface of the cells twice with PBS to aid in removing viral complexes. Complete removal is not necessary as no adverse effects on cell health have been observed from residual polymer complexes.
This fluorescence microscope image illustrates sufficient transduction of human sarcoma cells, stably expressing SLC seven A one with tropic virus when using fluorescent control vectors to monitor transduction efficiency. Quantification of fluorescence using fax analysis demonstrates a transduction efficiency of greater than 90%Transduction rates of HDFS are generally lower because the cells have not been blast aside in selected for receptor expression, tits of atropic lentivirus are generally 10 to 20%of VSV pseudo typed virus were measured on S-L-C-H-S-C. The MTT viability assay allows sensitive detection of growth arrest in target cells as seen here, transduction with virus plus concentrations of chondroitin sulfate.
Poly brain up to 800 micrograms per milliliter have no effect on HDF metabolism. Fax analysis of HDFS transduced with virus plus various concentrations of chondroitin sulfate. Poly green shows enhancement of transduction compared to poly green alone.
Maximum enhancement occurs at 100 micrograms per milliliter. Chondroitin sulfate poly brain several fold lower than previously reported values. Thus, it is important to optimize conditions for any given cell type complexation with chondroitin sulfate.
Poly brain enhances the observed titer, roughly threefold in S-L-C-H-S-C. In practice, this yields a greater effect on transduction efficiency at low virus concentrations than at higher concentrations, which is most likely due to multiple transduced cells and receptor saturation at higher virus concentrations. Transduction with tropic virus is specific for cells expressing neuron retrovirus receptor.
When transducing unmodified human cells with tropic virus fluorescence greater than the unreduced background has not been observed as seen by fax analysis microscopically. HDFS show no transduction in the absence of receptor while pret transduction with receptor results in fluorescent cells. When attempting this procedure, it is important to remember to verify that the virus will not transduce on modified human cells.
After watching this video, you should have a good understanding of how to use tropic lentivirus to deliver potentially hazardous genes to human cells, including the use of polymer complexation to enhance transduction.
