Evaluation of Abnormal Growth-related Genes of Hematopoietic Stem and Progenitor Cells by Combining CRISPR/Cas9 Technology with Cell Counting

The scope of research is leukemia pathogenesis. We aim to identify genes critical for abnormal HSPC proliferation by combining with CRISPR/Cas9 with CCK-8 assays, revealing their roles in tumorogenesis and the potential as drug targets.

CRISPR/Cas-9-based gene editing provide a simple and efficient approach for achieving gene knockout in your eukaryotic cells. The current experimental challenge is to further increase CRISPR/Cas-9 knockout efficiency.

Our protocol uses Cas-9 mice to supply bone marrow cells with consultative Cas-9 expression, thereby, viral delivery of SGIs into the cells, thereby, greatly improving experimental efficiency.

[Presenter] To begin, obtain the femur and tibia from the euthanized experimental mouse. Transfer the femur and tibia to a laminar flow hood immediately after removal to maintain sterility. Using a one-milliliter sterile syringe, flush the bone marrow cavity with PBS containing 2% FBS to collect bone marrow cells. Pipette the bone marrow cells up and down 30 to 50 times to disperse them into single cells. Transfer the cell mixture to a 50-milliliter tube and vortex to mix well. Add 10 microliters of the cell suspension to 40 microliters of erythrocyte lysis buffer, and place it on ice for five minutes to lyse the erythrocytes. Then add 50 microliters of Trypan blue solution. Mix well and count viable cells. Next, centrifuge the cells at 800 G for 15 minutes at four degrees Celsius and aspirate the supernatant. Then re-suspend the cells at a concentration of one to two times 10 to the power of six cells per milliliter in bone marrow, pre-stimulation medium. And seed 10 milliliters of the suspension into a 10-centimeter dish. Incubate the dish for 24 hours at 37 degrees Celsius. To collect the pre-stimulated cells the next day, add five to 10 milliliters of PBS containing 2% FBS and rinsing the plate vigorously. Transfer the cells to a fresh tube, and add Trypan blue to the cells. Count viable cells in the mixture. Next, centrifuge the cells at 800 G for 10 minutes at room temperature. After discarding the supernatant, re-suspend the cells in freshly prepared spinfection solution at a concentration of approximately 10 to the power of six cells per milliliter. Seed four milliliters of the suspension into each well of a six-well plate. Seal the plate with parafilm and centrifuge it at 1,500 G for 90 minutes at 32 degrees Celsius. After centrifugation, gently re-suspend the cells with a pipette without changing the medium. Incubate the plate for four to six hours. For the first transfection, aspirate approximately three milliliters of supernatant medium from each well into a tube, and add an equal volume of bone marrow pre-stimulation medium. If cells are accidentally aspirated, repeat the centrifugation and re-suspension steps as described earlier. For the secondary transfection, the next day, remove two to three milliliters of medium from each well and add an equal volume of freshly thawed retroviral stock solution along with appropriate amounts of hepas and polygreen. Centrifuge the plate at 1,500 G for 90 minutes at 37 degrees Celsius. After centrifugation, gently re-suspend the cells and continue incubating for four to six hours. Now, replace the medium as described earlier and incubate for 24 hours. Wash the plate with PBS containing 2% FBS and collect the cells. Filter the cells through a 70-micrometer sieve before counting the viable cells using Trypan blue. Next, centrifuge the cells at 800 G for 10 minutes at room temperature. Re-suspend the cells in DMEM containing 10% FBS and murine interleukin-3, interleukin-6, and stem cell factor. Centrifuge approximately 0.5 to 1x10 to the power of six cells at 800 G for five minutes and discard the supernatant. Store the cell pellet at -80 degrees Celsius for genomic DNA extraction. Now, add 1x10 to the power of four cells per well into a 96-well plate, making six replicate wells per group. Then add 100 microliters of sterile PBS to surrounding wells and incubate until different time points of 0, 24, 48, and 72 hours. Aspirate the medium at each time point, and add 110 microliters of medium containing 1/10th volume of CCK-8 solution to the wells. Incubate the mixture for one hour. Finally, measure the absorbance at 450 nanometers using a microplate spectrophotometer. Trp53 knockout significantly increased the proliferative capacity of mouse, hematopoietic stem and progenitor cells compared to controls at 24, 48, and 72 hours as measured by the CCK-8 assay. The Trp53 editing efficiency was estimated to be 7.3% using TIDE analysis. And next-generation sequencing revealed mutations occurring at different positions in the gene.