Our goal is to systematically identify and therapeutically target critical network hubs with a focus on non-coding RNAs in cancer. The current experimental challenge of our approach lies in the combinatorial capacity required to generate and screen large numbers of genes in parallel. We aim to pave the way for identifying novel combinatorial non-coding RNA targets or druggable structures to circumvent resistance and therapeutic escape.
In the future, we will translate this proof of concept tool into large scale combinatorial library screening efforts, potentially in combination with RNA binding proteins, both in vitro and in vivo. To begin count Lenti-X 293T HEK cells using Trypan blue dye in a hemocytometer. Plate 4 times 10 to the power of 6 Lenti-X 293T HEK cells per 10 centimeter plate and incubate to achieve approximately 80%confluency the next day.
From this step onwards, work in a class 2 laboratory. Mix 500 microliters of a suitable transfection medium with transfer vector, envelop plasmid, and pPAX2 vector. Incubate the mixture for five minutes at room temperature.
In a separate tube, mix 500 microliters of reduced serum medium with 36 microliters of PEI reagent at 1 milligram per milliliter concentration. Incubate the mixture for five minutes at room temperature. Combine the two mixtures and incubate for 15 minutes at room temperature.
Now carefully, dropwise, add the mixture to the cell medium containing six milliliters and incubate overnight at 37 degrees Celsius in 5%carbon dioxide. After 12 to 15 hours, discard the medium into an autoclaveable waste bottle using a serological pipette. Rinse consumables, including serological pipettes and pipette tips, with a disinfectant solution before disposing of them in the autoclave waste.
Add five milliliters of fresh DMEM to the transfected cells using a new pipette. To harvest lentiviruses, transfer the supernatant using a serological pipette to a 50 milliliter canonical tube, 48 hours post transfection. Then add 5 milliliters of fresh medium to the cells and incubate them for a further 24 hours.
Store the tube in the refrigerator. After incubation, repeat the collection of lentiviruses and pool it with the sample collected earlier. After the third harvest store combined supernatants of all three harvests at four degrees Celsius until filtration and centrifugation.
Centrifuge the combined supernatant for 5 minutes at 500G and 4 degrees Celsius in aerosol-tight containers and rotors approved for Risk Group 2 organisms, then filter the supernatant through 0.2 micrometer sterile cell culture grade syringe filters. Concentrate the filtered supernatant at 1000G and four degrees Celsius using a centrifugal filter unit with a 100 kilodalton molecular weight cutoff to approximately 500 microliters. Store virus aliquots of 50 to 100 microliters at minus 80 degrees Celsius until further use.
Plate 501-mel cells in a six well plate at 2 times 10 to the power of 5 cells per well with a final culture volume of 2 milliliters one day before transduction. Replace the culture medium with 2 milliliters of prewarmed DMEM supplemented with polybrene at 6 micrograms per milliliter final concentration. Work in a class 2 laboratory from this step onward.
Add 50 microliters of prepared SPD and SAD lentiviruses simultaneously to the cells dropwise. Shake the plate gently and incubate it at 37 degrees Celsius in a 5%carbon dioxide atmosphere. 16 hours post-transduction discard the medium into an autoclaveable bottle using a serological pipette.
Add 2 milliliters of fresh medium containing 10 micrograms per milliliter Blasticidin and 2 micrograms per milliliter puromycin. Repeat the medium replacement step after two days and handle transduced cells under S1 conditions after seven days post-transduction. Seed 0.1 times 10 to the power of 5 cells containing stable transfected SadCas9 KRAB and Sp-dCas9-KRAB seven days post transduction in a 96 well plate with 100 microliters of prewarmed DMEM.
On the day of transduction, replace the medium with 100 microliters of prewarmed DMEM supplemented with polybrene at 6 micrograms per milliliter. Add the appropriate volume of lentiviruses to the cells and incubate the plate. After 16 hours, replace the medium with fresh medium containing Zeoin, Blasticidin, and puromycin.
Perform luminescence detection five days post transduction using a cell viability assay according to the manufacturer's instructions. Western blood analysis confirmed successful expression of SpdCas9 and SadCas9 fusion proteins in transducer 501-mel cells. Quantitative polymerase chain reaction analysis showed effective knockdown of individual long non-coding RNAs RP11 and XLOC using specific guide RNAs, but only the XLOC-sa and RP11-sp pair successfully repressed both targets simultaneously.
Dual repression of RP11 and XLOC using the XLOC-sa and RP11-sp GRNA pair significantly reduced 501-mel cell viability to 59%compared to control.
