The overall goal of this procedure is to produce a genetic cross of the rodent malaria parasite plasmodium yoi in order to generate recombinant progeny. This is achieved by first injecting a group of mice with a mixed inoculum containing the two parental clones of the parasites to be crossed. Infected mice are then used to feed mosquitoes inside, which the gat of each parental strains will fuse into a diploid zygote myotic division at this stage can lead to genetic recombination, thereby producing recombinant progeny.
The resulting sporozoites are then harvested from the mosquitoes sali glands and re-injected into naive recipient mice in order to obtain blood stage parasites among which are individual recombinant clones that can be isolated by limited dilution. In a successful experiment, five to 10%of the cloned lines will be recombinants, which can be characterized by genotyping for appropriate genetic markers. This method can help answer key question in the malaria research field, such as what are the genetic determinants of drug resistant ants and transmissibility in malaria?
Parasites At room temperature for two vials containing the frozen blood stage, malaria parasites to be crossed. In this example, the two rodent malaria parasites strains used are plasmodium yoi, oi, and plasmodium. Yoi Nigerians fit a syringe with a 22 to 30 gauge needle and draw up 400 microliters of PBS pH 7.4 using the same syringe.
Draw up 100 microliters of the Thor infected blood, invert the syringe up and down until the content is homogeneously mixed. During this demonstration, anesthetized animals will be used in order to minimize pain and stress. Anesthetization is performed by injecting 50 microliters of anesthetic drug cocktail containing 82.5 milligrams per kilogram ketamine, and 7.5 milligrams per kilogram xylazine into the peritoneum of a mouse.
Monitor mice for depth of anesthesia and to prevent mortality. Using this procedure, mice will remain under anesthesia for 20 to 30 minutes. Before recovery, inject 200 microliters of parasite containing solution directly into the peritoneum of a mouse.
When finished, discarded the syringe in a sharp spin. Using this method, infect two mice with each of the two parasites drains to be crossed three to five days following the injections. Blood stage parasites will develop in the infected mice.
Clip mouse tail tip of the donor mice infected with the different parasite strains with sterile scissors. Collect a drop of mouse tail blood on a microscopic slide and perform thin smears. Stain the thin blood smear with guillem stain.
Guillem SA dye will not stain uninfected red blood cells due to the uninfected cells. Lack of a nucleus em SAI will stain parasite nucleus in the infected red blood cells parasites, red blood cells appear in the form of ring stages, trophozoites ski ons and rupturing maites. Once the paras anemia reaches one to 5%in both mice, blood stage parasites can be harvested for the mixed clone infection.
Clip mouse tail tip of the donor mice infected with the different parasites. Strains with sterile scissors as described earlier. Collect one microliter of mouse tail blood using a glass capillary in one milliliter of sterile PBS use 20 microliters of the blood suspension to analyze the red blood cell density with cell ome.
Using the red blood cell density and para measurements dilute the blood samples in a physiological citrate saline solution so as to obtain a concentration of infected red blood cells of 1 million per 100 microliters. Combine a sample of infected blood from the two donors in a one-to-one ratio to obtain a mixed clone inoculum. Next, inject 100 microliters of the mixed sample into the peritoneum of each mouse to be infected.
Use two mice for single clone infections to allow confirmation of the successful transmission of each parental strain. The sexual stages of the parasite called male and female gametocytes should begin to appear in the blood of mice around four days following the infection at that time point. Begin monitoring the mice daily for the appearance of gametocytes by collecting tail blood as described earlier and preparing em sustained thin blood tail smears.
Male and female cytes are recognizable due to the presence of large granules or pigments. The cytoplasm of male gato sites is pink, whereas the cytoplasm of female TER sites is blue. Only mature gato sites present a large vacuole.
Once mature cytes of both sexes are detected, the mice can be used to feed mosquitoes. Inject 50 microliters of ketamine, xylazine and anesthetic solution into the peritoneum of each infected mouse. Once the mice are anesthetized, place one face down on the top of a cage containing 50 to 100 female Anis defense enzyme mosquitoes that have been starved of sugar supply for 24 hours and allow the mosquitoes to feed for 30 minutes without interruption.
Humanely euthanize the mice immediately after the feeding period. If the transmission is successful, nine days after the feeding, the gametocytes taken up by the mosquitoes will have fused into diploid zygotes that mature into oocysts, which can be seen in the insects midgut. The more oocysts are detected, the higher the chances are that the recombinant parasites have been generated.
Use an aspirator to collect five to 10 infected mosquitoes from the cage and transfer the mosquitoes into a small container. Anesthetize the mosquitoes using carbon dioxide gas and transfer them into a glass Petri dish on ice. The male mosquitoes whose antennae are noticeably bushier than the females from be discarded.
Fill two syringes with PBS and fit them with 26 gauge half inch needles. Deposit about 100 microliters of PBS from a syringe onto a glass slide. Transfer five to 10 anesthetized female mosquitoes onto the drop of PBS and place the slide on a dissecting microscope using the needles.
Remove each mosquito's wings and legs and then holding the insect at the thorax and posterior abdomen. Pull the body apart until the midgut a white sack like body is released. Detach the midgut and discard the rest of the body while performing the dissections.
Be sure to keep the mid guts moist using the PBS from the dissection needles. Place a cover slip over the mid guts once the dissection is completed. Examine the mid guts under a light microscope at 100 times magnification.
OOS will be recognizable as thick wall circular structures that lie on the outer surface of the mid guts. Count the numbers of OOS in each midgut. 17 days after feeding sporozoites released from the sys should have migrated to the mosquitoes SIV glands, sporozoites of malaria parasites.
A thin spindle shaped cells to harvest sporozoites. First, prepare a collection tube using clean scissors. Cut off the lid of a small 500 microliter einor tube.
Heat the tip of a sterile 19 gauge needle and puncture the bottom of the tube. Fill one third of the tube with glass wall to create a filter. Insert the filter tube inside a larger 1.5 milliliter einor tube.
Once the filter apparatus is ready, collect and anesthetize the mosquitoes as described earlier. Air mount the insects for dissection as before, and remove the mosquitoes, heads, wings, legs and abdomens leaving. Only the thorax is containing the infected siv glands.
Using fine tipped forceps, transfer the thorax into a 1.5 milliliter homogenizer containing 500 microliters of PBS. Homogenize the thorax on ice to release the sporozoites from the saliva glands and using a glass pipette, transfer the homogenates into the filtration apparatus. Centrifuge the filter tube for 10 to 20 seconds at 1000 rotations per minute.
Once the homogenates have been spun down, collect the flow through using a syringe fitted with a 22 to 30 gauge needle. Inject 100 to 500 microliters of the sporozoite suspension into the peritoneum of a mouse. Begin monitoring the para and red blood cell density of the mice three days after infection.
Pick a mouse displaying a 0.1 to 1%para and whose infected red blood cells contain mostly single parasites as a donor for the cloning procedure. Withdraw an appropriate amount of blood in PBS dilute tail blood from the mouse in a cold one-to-one solution of calf, serum and mammalian ringer solution so as to attain a concentration of 0.6 to one infected red blood cell per 100 microliter and store the inoculum on ice. Clean the injection site on the tail with ethanol intravenously.
Inject 100 microliters of diluted infected blood in each of to 100 mice. In this manner, each individual mouse should receive either one or zero parasites. In a successful experiment, 20 to 50%of mice will show a para of 0.1 to 0.5%at day eight.
Five to seven days later, screen the mice for the presence of blood stage parasites by examining gem sustained thin blood smears. Progeny, parasites from the cross can then be genotyped by PCR of DNA, extracted from tail blood samples using genetic markers on different chromosomes such as microsatellites and single nucleotide polymorphisms. Parasites whose independent genetic loci are derived from the two parental clones are recombinant clones.
The blood from the animals infected with recombinant parasites can be cryopreserved for future use. In a successful experiment, five to 10%of the total mice will have independent recombinant progeny. After watching this video, you should have a good understanding of how to produce genetic process in malaria.
Parasite plus medium yoi. This method can be equally applicable to other species of parasites.
