The overall goal of this procedure is to measure the frequency of induced mutations in a reporter gene recovered from the germ cells of male transgenic mice following exposure to a chemical or physical mutagen. This is accomplished by first exposing male mice to a suspected germ cell mutagen. The second step is to collect the germ cells after they have progressed through the desired stages of spermatogenesis.
Next DNA containing the mutation reporting transgene is recovered from the germ cells. The final step is to package the recovered reporter genes into lambda phage particles. Ultimately, an in vitro positive selection assay is used to measure the proportion of reporter genes recovered from the germ cells that have been mutated.
The main advantage of this technique over existing methods, like the specific locus test or domino little test, is that mutations are measured directly in germ cells as opposed to scoring mutant offspring. A large number of germ cells can be investigated from a single male, improving the sensitivity of the assay over traditional methods, while at the same time dramatically reducing the number of animals resources and time. This method can address key issues in the field of genetic toxicology and mutagenesis, such as the identification of agents that are mutagenic to germ cells and the elucidation of their modes of action.
Although we describe the method for the Muta mouse model, it can be easily applied to other transgenic rodent systems that carry bacteria phage mutation reporting vectors. Furthermore, while the method describes the process for male germ cells, it has general applicability to somatic tissues, and importantly, it is covered by an internationally harmonized test guideline published by the OECD. Generally, individuals new to this method will struggle with the timing of the in vitro portion of the assay because several of the steps have long waiting periods.
Steps must be carefully planned and coordinated to complete this method in a timely fashion. Demonstrating the procedure will be John Gingrich and Linda Soper biologist in my laboratory Prior to beginning the experiment. Three critical experimental variables should be selected, the administration time, sampling time, and the collected germ cell population.
These variables can be adjusted to interrogate the effects of exposure in various germ cell types and at different phases of spermatogenesis. To begin this procedure, randomly distribute eight to 12 week old transgenic male mice into a control group and treatment groups with a minimum of five per group. Then treat the mice with a test compound and a relevant control via an appropriate exposure route for the selected administration time.
Next, choose the appropriate sampling time according to the sperm metagenic cell type of interest. Then after the sampling time has occurred, euthanize the mice by cervical dislocation under isof fluorine anesthesia. To remove the testes, make an incision in the abdomen, locate the epidermal fat pad.
Carefully pull the fat pad to draw the testes and epididymus from the scrotum. Then excise the cota epididymus. Freeze the Cota epididymus in liquid nitrogen and store at minus 80 degrees Celsius for later use.
When ready to process the Cota sperm, defrost the Cota epididymus on ice and then transfer the thawed cota to a Petri dish. Next, use a scalpel or razor blade to thoroughly mince the coda Epididymus pipette 700 microliters of room temperature DPBS into the Petri dish. Then use a wide bore 1000 microliter pipette tip to release the sperm from the coda by drawing and releasing the suspension approximately 10 times or until the DPBS becomes cloudy with sperm, filter the sperm suspension through a stainless steel mesh filter into a fresh 1.5 milliliter tube, and then centrifuge the tube at 11, 000 times G for three minutes.
Carefully decant the supernatant out of the tube without disturbing the pellet. Then add one milliliter of cold, one x saline sodium citrate, and vortex the tube until the pellet is completely resuspended. Next, next, add 15 microliters of 10%SDS to the tube and vigorously shake the tube for 30 seconds to disrupt the non sperm cells.
Then centrifuge the tube at 11, 000 times G for two minutes. After centrifugation, carefully decant the supernatant out of the tube and add 940 microliters of 0.2 x cold saline sodium citrate, and vortex the tube again until the pellet is resuspended. Add 120 microliters A beta mer capto ethanol 100 microliters of 10%SDS 20 microliters of 0.5 molar EDTA pH eight and 20 microliters of 60 milligrams per milliliter.
Proteinase K to the resuspended sperm. Mix the solution well and then place the tube on a rotator overnight at 37 degrees Celsius to digest after the overnight digestion. Extract the DNA from the coda sperm by following the steps outlined in the text protocol for performing a phenol chloroform extraction after extraction.
Dissolve the DNA precipitate in 40 to 100 microliters of tris EDTA buffer PH eight and store at four degrees Celsius. Allow several days for the DNA to fully dissolve before proceeding to the next step on the day. Prior to performing the LAX Z mutation assay, prepare and autoclave a sufficient amount of bottom auger for the number of samples being processed.
Then aseptically, pour the auger into 90 millimeter Petri dishes and allow the auger to solidify in a 50 milliliter tube. Add 10 milliliters of LB broth, 100 microliters of 20%maltose solution, 25 microliters of 20 milligrams per milliliter ampicillin, and 20 microliters of five milligrams per milliliter. Can mycin then inoculate the tube with LAX Z negative GAL e negative e coli, and grow overnight at 37 degrees Celsius while shaking at 240 RPM on day one of the assay subculture the cells by preparing a one to 100 dilution of the overnight culture in fresh LB with no antibiotics.
Incubate the culture at 37 degrees Celsius with shaking at 240 RPM for about three hours or until the OD 600 equals one. Once the OD 600 reaches one, divide the cell suspension evenly into 50 milliliter tubes and centrifuge at 1, 300 times G at 15 degrees Celsius for 10 minutes. Then remove the supernatant and resuspend the cells in half of the original volume of LB containing 10 millimolar magnesium sulfate.
Put the cells aside while the other reagents are prepared. Package the DNA in Lambda phage particles by first pipetting. Four microliters of the DNA extracted from the coda sperm using a wide bore pipette into a 1.5 milliliter tube.
Defrost the first tube from a phage packaging extract kit at room temperature, and then pipette 4.8 microliters of the packaging extract into the tube containing the DNA. Next, gently stir the solution with the pipette tip and then incubate the tube in a 30 degree Celsius water bath for 1.5 hours. Next, defrost the second packaging extract tube and again, pipette 4.8 microliters into the tube containing the DNA.
Gently stir the solution with the pipette tip and then incubate the tube in a 30 degree Celsius water bath for 1.5 hours. Resuspend the packaged phage particles in 500 microliters of saline magnesium buffer, and place the tubes on a rotator for 30 minutes at 20 RPM. After rotating, briefly vortex the samples and then centrifuge at 11, 000 times G for 30 seconds to collect the samples at the bottom of the tube.
The phage particles are now ready for infection. Next, prepare eight milliliters of top auger for each titer and mutant plate. Maintain the top augers at 50 degrees Celsius prior to adding magnesium sulfate to 10 millimolar and add three grams per liter of the selective agent PGAL to the mutant selection.
Top auger only prior to infection of cells. Label two 50 milliliter tubes per sample label one mutant and one titer. Then label eight auger plates per sample, four mutant and four titer.
Next, pipette two milliliters of resuspended cells into each 50 milliliter tube. Add 500 microliters a packaged phage particles into a 50 milliliter tube labeled mutant. Gently mix the tube and allow the phage particles to infect the cells for 30 minutes at room temperature.
After the 30 minute incubation, briefly vortex the infected cells and then transfer 15 microliters of the infected cells to the corresponding 50 milliliter titer tube. Add 30 milliliters of 50 degrees Celsius titer top auger to the titer 50 milliliter tube. Immediately distribute the auger and cell mixture among the four titer plates.
Ensure that three grams per liter P gal has been added to the 50 degree Celsius mutant selection auger. Next, add 30 milliliters of the mutant selection auger containing PGAL to the mutant 50 milliliter tube. Then immediately distribute the auger and cell mixture amount to the four mutant plates.
Allow the plates to solidify and then invert the plates and incubate at 37 degrees Celsius overnight. After the overnight incubation, count the number of plaques on the mutant and titer plates. Then estimate the mutant frequency by dividing the total number of mutant plaques counted, counted on the four mutant plates by the estimated total number of plaque forming units in the total volume of infected cells determined from the titer plates.
A typical transgenic rodent germ cell mutation assay results in very few plaques on the mutant auger plates, especially in the control dose groups. While on the other hand, hundreds of plaques are detected on the titer plates. Exposure of mutt mouse males to a single acute oral dose of 0 25, 50, and 100 milligrams per kilogram ENU followed by a 70 day sampling time permitted the measurement of the mutational events in spermatogonia stem cells.
The low dose of ENU induced a 2.6 fold increase in mutant frequency above controls, and the maximum dose elicited a 4.4 fold increase. While designing experiments that use this procedure, it's important to always take into account the timing of it. Sperm metagenic cycle, the administration time, sampling time, and the collected cell type must be carefully selected in order to observe effects originating from the desired sperm metagenic cell type.
Following This procedure, DNA sequencing can be performed on the mutant plaques to characterize the mutations and identify mutants that may be derived from clonal expansion events. This technique paves the way for researchers in the field of genetic toxicology to identify mutagens that may target the germline. After watching this video, you should have a good understanding of how to determine the induced mutant frequency in the germ cells or transgenic male mice exposed to a mutagen.
This assay has potential applications in regulatory testing for gem cell mutagens, and in more targeted mechanistic investigations.
