For lentiviral infection, plate the trypsinized MPNST target cells after counting them, maintaining 2.5 million cells per 15 centimeter dish. Transduce the cells with the thawed virus at a 0.5 multiplicity of infection, in the presence of five micrograms per milliliter of the cationic polymer. Trypsinize the cells three days after the addition of puromycin.
Centrifuge half of the cell population at 200 G for five minutes in a tabletop centrifuge. After re-plating the other half of the cell population, grow them for approximately seven population doublings before harvesting and centrifuging as previously demonstrated. Divide the re-suspended cell pellet corresponding to the desired time into two 15 milliliter polymethylpentene tubes.
Add 500 microliters of 10%SDS per tube. Mix and incubate at room temperature for five minutes. Place the tubes in a DNA shearing device to sonicate the DNA.
Following the addition of phenol and chloroform. Mix well by vortexing vigorously at the maximum possible speed for 45 to 60 seconds. Transfer three milliliters of the resulting clear upper phase from each tube to another fresh 15 milliliter tube.
Add 0.5 milliliters of three molar sodium acetate and four milliliters of isopropanol and mix well. After centrifuging the tubes for 30 minutes at 7, 200 G and 20 degrees Celsius and discarding the supernatant, add 0.5 milliliters of 70%ethanol to the pellet and dislodge it by pipetting up and down. Combine the re-suspended pellets from both tubes into a 1.5 milliliter centrifuge tube.
Perform the PCR reactions using the conditions shown on the screen. After the second PCR reaction, combine the four samples into one micro-centrifuge tube. And add 80 microliters of 6X loading dye to it.
Visualize the gel and confirm a band at approximately 250 base pairs. Human MPNST cells transduced with a non-target control and lentivirus, expressing four different Bcl-6 short hairpin RNA, revealed that several of the Bcl-6 short hairpin RNAs markedly reduced cell numbers. The immuno blot showed that the degree of decrease in cell numbers correlated with the degree of Bcl-6 knockdown.
