The overall goal of this procedure is to carry out a PCR based technique to quantify donor cell engraftment in a competitive murine bone marrow transplantation model, this is accomplished by first performing bone marrow transplantation in female recipient mice using male donor cells. Next peripheral blood is collected from recipient mice at different time points and the genomic DNA is is isolated. The samples are prepared for a standard curve.
Finally, real-time PCR is carried out with standard samples and test samples. Ultimately, results can be obtained that show the engraftment of male donor derived cells in the female recipient mice through calculation against a standard curve with a known percentage of male versus female DNA. The male advantage of this technique over existing method like a flow cytometry based method, is that it is more sensitive, more economic and samples can be stored for a long time.
Mostly importantly, our technique is most string independent and can be used for any genomic background of mice like well defined cell surface marker to differentiate the donor cell from recipient cells After euthanizing FVB nj, male donor and female mice under a laminar flow hood use small scissors and forceps to dissect out the femur and tibia bones and place them in a 60 millimeter tissue culture dish containing six milliliters of ice cold RPMI 1640 with 5%heat inactivated. FBS use a Kim wipe to remove muscle and other tissues and cut off both ends of each bone shaft in the dish. Then connect the end of the bones to a 23 gauge needle attached to a three cc syringe filled with RPMI 1640 with 5%heat inactivated FBS and flush out the marrow.
Use the same needle to disaggregate the bone marrow tissue by repeated aspirations. Transfer the cell suspension to a 15 milliliter centrifuge tube and spin it down for five minutes at 400 G.After removing the supernat, resuspend the cells in one milliliter of room temperature, red blood cell lysis, buffer, and incubate at room temperature for five minutes before adding five to 10 milliliters of RPMI 1640 with 5%heat inactivated FBS pass the cells through a cell strainer and collect the flow through into a new tube. After spinning down the cells for five minutes at 400 G and removing the supernatant, the pellet should be absent of red color indicating the full removal of red blood cells resuspended in 10 milliliters of RPMI 1640 with 5%heat inactivated FBS after gently vortexing, add a small aliquot to a hemo cytometer and count the cells.
Calculate the volume of cells required to mix with competitor female cells at a ratio of five to two for bone marrow transplantation. As a final step to prepare cells for injection, mix the donor and competitor cells pellet the cells wash with PBS and resuspend in PBS at a final concentration of five times 10 to the sixth per milliliter donor cells and two times 10 to the sixth per milliliter for competitor cells, four to six hours before bone marrow transplantation. Irradiate female recipient mice with cesium 1 37 gamma radiation at a single dose of 11 grays.
Place the irradiated female mice in a mouse restrainer and inject the mixed donor and competitor cells via the tail vein in a 0.1 milliliter total volume such that each mouse receives five times 10 to the fifth donor cells and two times 10 to the fifth competitor bone marrow cells to draw blood samples. After anesthetizing, the mice collect about 50 microliters of blood into EDTA coated tubes by retroorbital bleeding. Also collect samples from age-matched male and female mice.
Next to isolate genomic DNA from subject mice and mice. For the standard curve, add about 200 microliters of room temperature RBC lysis buffer to each blood sample and incubate for five minutes. Add one milliliter of PBS and spin down to remove most of the lysed RBCs.
Then use a blood DNA extraction kit to isolate the DNA pre warming the elucian buffer to 37 degrees Celsius. To enhance the yield measure the DNA concentration of each sample. Those with an OD two 60 to two 80 ratio between 1.8 to 2.0 are used for further analysis.
To set up a standard curve, dilute the male and female DNA to a concentration of four nanograms per microliter and prepare the DNA mixture according to these guidelines. Set up the PCR reaction plate by mixing cyber green super mix reagent with the primers shown here and genomic DNA. Each 20 microliter reaction contains 400 nano molar of each primer and five microliters of blood cell genomic DNA run in triplicate.
Carry out PCR according to the conditions found in the text protocol and obtain the cycle threshold or CT values. Calculate delta CT times the quantity CT to the power of ZY one minus CT to the power of BCL two and the ZY one expression levels or two to the power of minus delta CT generate standard curves by ploting the mean of the triplicate two to the power of delta CT values to the known percent male DNA in the mixture with a linear regression fitting shown. Here are examples of standard curves plotted with mean values of two to the power of delta CT against percentages of male DNA.
This plot includes a specific melting temperature for BCL two and ZFY one amplicons localized at 78.5 and 88.5 degrees Celsius respectively. BCL two is used as a reference gene to normalize the total amount of loaded DNA in each PCR reaction. BCL two amplification curves for each standard sample merge with each other independent of male DNA concentration indicating equal amounts of loaded DNA.
However, ZFY one amplification curves migrated to the left with increasing amounts of male DNA in the sample hematopoietic stem cells from PIM TKO mice have defects in reconstituting lethally irradiated mice, and as expected recipient mice transplanted with PIM TKO cells had a much lower percentage of male cells compared to mice transplanted with wild type cells. To validate our method, we tested male donor cell engraftment in transplanted female mice, receiving bone marrow cells from wild type versus PIM triple knockout or TKO mice at different time points post transplantation, both peripheral blood at six weeks and bone marrow samples at 16 weeks. Were analyzed After watching this video.
You should have a good understanding of how to use PCR based method to quantify the donor cell engraftment in a competitive marine bone marrow transplantation model.
