A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

The overall goal is to report a novel gene transfer technique for differentiation of osteoclast in vivo. We also describe cell culture protocols for differentiating mouse's bone marrow macrophages of human pbmc into osteoclast. This video will be broken into three parts.

One, hydrodynamic delivery of rank lichen mini circle, DNA, two in vitro osteoclast generation from mouse, bone marrow macrophages, and three in vitro osteoclast generation from human pbmc. Part one, hydrodynamic delivery of rank lichen mini circle, DNA. The following animation shows a brief overview of hydrodynamic delivery via mouse tail vein injection.

First place mouse under a heat map for 10 minutes prior to injection. This dilates the blood vessels and makes the lateral veins more visible. Then locate the injection site and proceed with the injection, making sure to complete the injection within five to seven seconds.

The next portion of this video will show a more detailed account to the procedures involved in the hydrodynamic delivery via mouse tail vein. Gather the materials for an injection. Warm up the mouse in a cage for 10 minutes prior to injection.

Monitor mouse carefully to avoid dehydration and hypothermia. Warming the mouse dilates the blood vessels and makes the lateral veins more visible. Once the veins are visible, transfer the mouse to a restrainer and insert a plunger to restrain mouse movement.

Disinfect injection site with an alcohol wipe. Using a 27 to 30 gauge needle for the mouse tail injection with diluted rank ligand or GFP mini circle into Prewarm ringer solution. Hold the tail firmly with one hand and insert the needle into the tail vein.

Apply pressure on the syringe and complete the injection within five to seven seconds. Afterwards, remove the needle from the tail of vein and apply pressure with the cotton ball onto the injection site to stop any bleeding. Increased breathing rate due to diluted blood should be observed and successful injections.

Monitor mouths for 15 to 30 minutes, then transfer back to the barium. Once the mouse cuisines normal breathing rate, hydrodynamic gene delivery utilizes hydrodynamic pressure generated by a rapid injection of large volume fluid into a blood vessel to overcome the physical barriers of endothelium cell membrane that prevents DNA from entering parenchyma cells. Parenchyma cells are transfected because the proximity to capillary endothelium allows for immediate access of DNA.

Once the endothelial barrier is disrupted. Part two, in vitro osteoclast generation from mouse bone marrow macrophages, the first step is to sacrifice the mouse isolate tibia and femur bones. Flush out bone marrow using a syringe loaded with PBS, then count cells and then plate briefly.

After euthanizing the donor mouse and removing the femur and tibia bones, the bone marrow is flushed into a 50 milliliter tube. Using a one milliliter syringe with a 25 gauge needle loaded with PBS mix and then transfer bone marrow suspension to a new 50 milliliter tube by passing it through a nylon cell strainer. After passing the bone marrow suspension through a nylon cell strainer centrifuge down the cells and resus suspend in osteoclast culture medium, prepare a 96 well plate using five millimeter dentine slices.

Similarly, you can prepare a 96 well plate using five millimeter glass cover slips. Notice the place dentine slice on the left and the glass cover slip on the right using a hemo, cytometer, or automated cell counter count the cells and then plate 300, 000 cells to glass cover slips or denting slices placed in the 96 well plate and incubate in a 37 degrees Celsius incubator. Remove all media containing all non-adherent cells and add new also class medium supplemented with MCSF for 48 hours.

After 48 hours, remove all media and then add osteoclast media containing mouse MCSF and mouse rank lain to initiate osteoclastogenesis, remove and replenish media every three days as required. Mature osteo class defined by the formation of multinucleated trap positive cells. F active formation and resorption on dentine part three.

In vitro also class generation from human pbmc. Add 10 milliliters of prewarm histo pack into a 50 milliliter tube am the leukocyte filter obtained from a blood bank flushing with PBS into a 50 milliliter tube. Then layered the blood over the histo pack solution very slowly, making sure that the blood does not mix with the histo pack solution.

Note how the blood is layered on top of the histo pack solution centrifuge samples at 2, 250 RPM for 20 minutes at 18 degrees Celsius. Notice the Buffy coat layer in the center of the three layers. After centrifugation, isolate the white buffy coat layer containing the white blood cells and transfer to a new 50 milliliter tube.

Then after the completion of the step, dilute the cells with PBS and centrifuge again at 1800 RPM for five minutes to collect the cells. After centrifugation, remove the super name. Re suspend the cells in osteoclast culture media mix and agitate the cells and media together.

Count cells with hemo, cytometer or cell counter Prepare a 96 well plate with denting slices or glass cover slips as previously demonstrated in this video. Then plate 1 million cells per well on glass cover slips or denting slices 24 hours after plating. Add human MCSF into the culture media.

Change culture media, replenishing the cells with human MCSF every two to three days as required. Then proceed to the differentiation step by adding human rank ligand supplemented with human MCSF and osteoclast media to initiate osteoclastogenesis, mature osteoclast defined by formation of multinucleate trap positive cells, F actin ring formation and resorption on dentine results. Here we describe our in vivo osteoclastogenesis mouse model using hydrodynamic delivery of soluble rank ligand mini circle, DNA, as well as in vitro culturing and differentiation of precursor cells.

To osteoclast. Figure one shows the successful gene transfer of GFP and rank ligand mini circle in mice, figure two shows representative images of mouse bone marrow or PBMC cell differentiation time course cultures of precursor cells to osteoclast by brightfield Microscopy figure three shows representative images from three independent methods for characterizing mouse bone marrow derived osteoclast. We demonstrate that the presence of soluble rank ligand activates the expression and release of trap figure three A and B, the formation of F actin rings.

Figure three C and D.We also show by scanning electron microscopy that soluble rank lichen induces the formation of resorption pits on dentine figure three E and F.Figure four shows the same characterization methods on PBMC derived osteoclast. Again, the presence of soluble rank ligand activates the expression and release of trap figure four A and B.The formation of F actin rings Figure four C and D and soluble rank ligand induces formation of resorption pits on dentine figure four E and F.Together these techniques form powerful investigative tools for osteo class differentiation and activation. Using these systems, we are able to generate osteo class in vivo and in vitro and define the stimuli and signals required for proliferation and activation, as well as test the efficacy of pharmacological and biological inhibitors.