We've developed an efficient and economical method to establish mice with gene manipulation specifically in hematopoietic stem cells, Using genome editing technology and lentivirus-mediated transgene delivery systems. The advantage of this protocol is that we can create animal models harboring specific mutations in hematopoietic cells in a rapid and cost-effective manner compared to conventional mouse transgenic approaches. Demonstrating the procedure will be Ying Wang, a researcher from my laboratory.
To generate lentivirus particles, start by coding a six well plate with collagen solution and incubating it at 37 degrees Celsius and 5%carbon dioxide for 30 minutes. Then, seed 293T cells onto the plate and incubate it for another two hours. Meanwhile, prepare the mixture of three transfection plasmids by combining 0.9 micrograms of lentivirus vector, 0.6 micrograms of psPAX2 and 0.3 micrograms of pMD2.
G and deionized to 10 microliters per well. Then, carefully add 50 microliters of 1X PBS and five microliters of diluted PEI MAX to the plasmid mixture, and incubate the mixture at room temperature for 15 minutes. After the incubation, add one milliliter of DMEM.
Aspirate the media from the six well plate and add one milliliter of plasmid mixture to each well. Incubate the plate for another three hours. Replace the media with fresh DMEM and incubate for 24 hours.
Add one milliliter of fresh DMEM and incubate an additional 24 hours. After total 48 hour incubation, transfer the supernatant to a 50 milliliter tube and centrifuge it at 3000 times G for 15 minutes to remove any free-floating cells. Filter the supernatant through a 0.45 micrometer filter and ultra-centrifuge it for three hours.
Carefully aspirate the supernatant, leaving behind the white pellet. Re-suspend the pellet with 100 microliters of serum-free hematopoietic cell expansion medium without aeration. Keep a 10 microliter aliquot to measure the viral titer and store the rest of the suspension at minus 80 Celsius until ready to use.
After isolating the bones, place them into a 50 milliliter conical tube with RPMI and place the tube on ice. In a bio-safety cabinet, transfer the bones into a sterile 100 millimeter culture dish. Then grasp a bone with blunt forceps, and use dissection scissors to carefully cut both epiphyses.
Fill a 10 milliliter syringe with ice cold RPMI and use a 22 gauge needle to flush the bone marrow from the shaft into a new 100 millimeter culture dish. After all the bone marrow has been collected, make a single cell suspension by passing the bone marrow through a 10 milliliter syringe with an 18 gauge needle. Filter the cell suspension through a 70 micrometer cell strainer into a 50 milliliter conical tube.
Then centrifuge the tube according to the manuscript directions and aspirate the supernatant. Re-suspend the cell pellets in an appropriate volume of optimized separation buffer. To isolate lineage negative cells, use a lineage cell depletion kit according to the manufacturer's instructions.
Re-suspend the isolated lineage negative cells in serum-free hematopoietic cell expansion medium. Pre-incubate the cells at 37 degrees celsius in 5%carbon dioxide for two hours. Then add lentivirus according to the manuscript directions and leave the cells in the incubator for another 16 to 20 hours.
The next day, collect the lentivirus-transduced cells in a 15 milliliter conical tube and centrifuge them at 300 times G for 10 minutes. According to NHA guidelines, it is important to handle lentivirus vectors at Reece Group level two. Precautions must be taken, based on facility regulations.
Carefully aspirate the supernatant and re-suspend the pellet in 200 microliters of RPMI per mouse. Keep the cells at room temperature until transplantation into mice. After the second irradiation session, retro-orbitally inject transduced delineage negative cells into each anesthetized mouse using an insulin syringe.
Three to four weeks after bone marrow transplantation, obtain blood samples from the retro-orbital vein using capillary tubes. Transfer 20 microliters of blood into round-bottom polystyrene test tubes and place on ice. After RBC lysis, incubate the cells with a cocktail of monoclonal antibodies in the dark for 20 minutes at room temperature.
After incubation, wash, fix and centrifuge the cells and re-suspend the cell pellet in 400 microliters of FACS buffer. Keep the samples at four degrees Celsius until analysis by flow cytometry. Success of lineage negative cell transduction can be evaluated with flow cytometric detection of RFP.
It has been demonstrated that after seven days of in vitro culture, on average, 75.7%of cells are transduced. Flow cytometry has also been used to analyze mouse peripheral blood after reconstitution with transduced and non-transduced hematopoietic stem cells. An average of 94.8%of neutrophils, 93.5%of monocytes, and 82.7%of B cells express RFP.
Genomic DNA from the RFP-positive blood cells has been PCR amplified and sub-cloned for sequence analysis. Various mutations are detected and 69%of the clones show out of frame or premature stop mutations. To succeed with this procedure, preparation of high antivirus stock and optimized conditions for transduction and transplantation of hematopoietic cells are required.
We have applied this technique to study the law of in cardio-vascular disease. But it is also useful to study hematological malignancy. This method enables us to study the functions of new types of genes in hematopoietic system in high efficient and high manner.
We gather this over the availability of transgenic mice.
