A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The overall goal of this procedure is to identify and validate drug resistant mutations. This is accomplished by first creating a random mutation library of the targeted oncogene. The second step is to perform in vitro screening for the emergence of resistant clones.

The third step of the procedure is to sequence the resistant clones to identify resistant mutations. The final step is to validate the resistant mutations by recreating the mutations using site directed mutagenesis, followed by in vitro and in vivo assays. Ultimately clinically relevant drug resistant mutations can be identified against a given drug target pair.

Main advantage of this technique over existing methods like ENU or PCR based methods is that it's unbiased and unlike PCR based METAGENE ssis, this one is not limited to a size of the oncogene or the GC content of any gene. Generally, those new to this technique will struggle due to a lack of knowledge in molecular and cell biology. Visual demonstration of this method is critical.

As neogenesis and screening steps are difficult to learn. It's because many people lack experience in viral production, in cell culture and semi cell media. This procedure begins with the transformation of XL one red e coli cells with a plasmid bearing JAK two V 617 f an oncogenic isoform of JAK two XL one red e coli cells are defective in DNA repair mechanisms, thus allowing them to incorporate random mutations during replication.

Mix 50 to 100 nanograms of plasmid DNA with 100 microliters of competent cells in a pre chilled polypropylene tube. Do not use more than 100 nanograms of DNA because it decreases the transformation efficiency. Incubate on ice for 30 minutes, gently swirling every five minutes for good library coverage.

Use four to six tubes of competent cells. Immerse the polypropylene tubes in a water bath at 42 degrees Celsius for a heat shock of 45 seconds, and then incubate on ice for two minutes. Next, add one milliliter of SOC medium to each tube.

Incubate the tubes at 37 degrees Celsius while shaking at 225 to 250 RPM for 90 minutes. Plate cells on four 10 centimeter LB auger plates containing 100 micrograms per milliliter of ampicillin. Incubate the plates at 37 degrees Celsius for 16 to 24 hours.

Once colonies are visible, collect them by scraping the plates with a sterile plate scraper. Pool the cells from each plate. Subsequently, the plasmid DNA is isolated using a commercial plasmid extraction kit and the heterogeneity of mutations in the library is assessed by sequencing one day before transfection plate, four times 10 to the sixth HEK 2 93 T cells, onto 100 millimeter dishes in DM EM containing 10%FCS, penicillin, streptomycin, and two millimolar L-glutamine.

On the following day, prepare the transfection reagents for each 100 millimeter dish. Mix five micrograms of the plasmid library, five micrograms of a retroviral packaging plasmid and 40 microliters of FU gene to a total volume of 400 microliters using serum free DM E EM media. Incubate the DNA and lipid mix for 20 minutes at room temperature.

Next, replace the medium of the HEK 2 93 T cells with fresh medium and add the DNA lipid complex dropwise on top of the cells. Incubate at 37 degrees Celsius for six hours. After six hours, replace the transfection media with fresh media and incubate the plates for 48 hours at 37 degrees Celsius per virus production.

After 48 hours of incubation, collect viral supernatants filtered through a 0.45 micrometer acro disc filter to begin the procedure for screening and identifying drug resistant JAK two V 607 F mutants transduce BAF three cells with the retroviral supernatants. Mix 100 million BAF three cells with 100 milliliters of virus supernatant to a total volume of 300 milliliters using RPMI media containing 10%serum and poly brain. Distribute this cell and virus supernat mixture to multiple six well plates filling each well with four to five milliliters.

Centrifuge the plates at 1, 250 GS for 90 minutes at 37 degrees Celsius. After centrifugation, incubate the plates at 37 degrees Celsius for 24 hours on the following day, pool the transduced cells for each drug concentration. Mix 10 milliliters of cells with ruxolitinib in a 50 milliliter tube.

The concentrations of ruxolitinib tested in this example are 1 5 2 0.5, and 10 micromolar. Make up the volume to 40 milliliters using RPMI containing 20%serum. Add 10 milliliters of 1.2%auger to the cells and mix carefully plate in six well plates.

Dispensing four milliliters per well. Incubate the plates at 37 degrees Celsius for two weeks. Once resistant colonies are visible, use 0.2 milliliter pipette tips to pick the colonies and subculture them individually.

In 24 well plates for three to four days harvest the resistant BAF three cells by centrifugation at 450 GS for five minutes at room temperature. After isolating the genomic DNA using a commercial genomic, DNA extraction kit, amplify the full length JAK two CDNA from the genomic DNA by PCR. The PCR products are then separated by Agar Rose's gel electrophoresis and the 3.4 KILOBASE DNA band.

Representing the JAK two coating frame is isolated using a commercial gel extraction kit. Finally sequence the full length CDNA using eight internal primers spanning the coding sequence and analyze the sequences using commercial software to validate drug resistant mutations. In vitro selected variants are generated by site directed mutagenesis of the JAK two V 617 F sequence and reintroduced into BAF three cells.

Add 50 microliters of media containing various concentrations of ruxolitinib to the wells across the plate separately, plate 10 to the fourth of the BAF three JAK two V 617 F cells into each well of a 96 Well plate incubate the plates at 37 degrees Celsius for 60 hours To assess cell viability, add 10 microliters of WST one reagent to each well and incubate at 37 degrees Celsius for two to four hours. Record the absorbance at a 450 using a plate reader. Perform all assays in triplicate and plot the average absorbance against ruxolitinib concentrations.

Perform a best fit sigmoidal curve using a non-linear curve fitting algorithm score. The drug concentration resulting in 50%cell viability as the cellular IC 50 subsequently in vivo validation of resistant mutations is performed as described in the accompanying text protocol. Since the resistant cell clones generated by random mutagenesis may carry more than one mutation, these mutations must be validated in isolation by both in vitro and in vivo assays.

In the in vitro assay, selected variants were generated by site directed mutagenesis of the JAK two V 607 F sequence and reintroduced into BAF three cells, which were then measured for cell proliferation ability at different dosages of ruxolitinib to evaluate IC 50 values. In addition, immuno blotting for phosphotyrosine or phospho STAT five was performed on protein lysates of BF three cells that were incubated with increasing doses of ruxolitinib to rule out any off target mediated resistance. Mutants exhibiting enhanced IC 50 values and persistent auto phosphorylation at higher ruxolitinib concentrations are thus confirmed to be drug resistant variants to validate in vivo resistance.

BF three cells engineered to express JAK two V six 17 F variants and luciferase cherry were injected into mice which were subsequently injected with ruxolitinib for two weeks. After two weeks. The mice were imaged for luciferase catalyzed bioluminescence, BAF three chimerism in bone marrow and peripheral blood flow was assessed by measuring the fluorescence of cherry positive cells.

The results show that mice expressing JAK two V 607 F are to ruxolitinib while resistant variants show progressive increments in bioluminescence over the period of treatment. Once mastered, random neogenesis can be performed in four to five days if it's done properly. After watching this video, you should have a good understanding of how to develop a randomly ized plasmid, DNA library for in vitro screening against any drug target pair.

Don't forget that working with retroviruses can be extremely hazardous. Precautions shall always be taken while performing this procedure.