Currently we have a limited understanding of the biology of plasmodium infection of the liver. This protocol has helped us generate a lot of transgenic lines that has helped answer some critical questions about the biology of the organism. A fundamental understanding of the parasite and its biology are required to develop new tools and approaches to combat the infection of malaria.
The method of transgenesis that we are describing here has helped us decipher some key and complex biology of the organism as well as the host. Demonstrating this procedure will be Carson Bowers, my laboratory manager. After generating P.Bergehei infected mice and euthanizing them, collect two milliliters of blood from two infected mice into five milliliters of Elsevier solution in a sterile environment.
Centrifuge the blood at 450 G for eight minutes at room temperature. Discard the supernatant and re-suspend the cell pellet in 10 milliliters of complete RPMI media. Centrifuge the cell suspension again and re-suspend the pellet in 25 milliliters of complete RPMI media.
Transfer the cell suspension to a T75 culture flask with a filter cap and incubate with gentle shaking to keep the cells in suspension. At the end of the incubation, collect 500 microliters of the sample. Centrifuge and re-suspend the pellet in 10 microliters of complete RPMI media.
Next, prepare a thin blood smear. Stain the blood smear with Giemsa stain and determine the frequency of schizonts. For optimal transfection efficiency, 60 to 70%of the parasites should be in the schizont stage.
Prepare a gradient centrifugation buffer by reconstituting 13 milliliters of density gradient stock medium in a 50 milliliter centrifugation tube. Transfer 25 milliliters of blood culture to a fresh 50 milliliter centrifugation tube. Then carefully layer 20 milliliters of gradient centrifugation buffer to the bottom of the centrifugation tube, using a narrow glass pipette to create a gradient column and ensure a clear division between the buffer and the media.
Centrifuge the prepared buffer and media for 20 minutes at 450 G with no break at room temperature. Using a pasture pipette, carefully remove the schizont layer at the interface of the culture media and the gradient centrifugation buffer. Place it in a new 50 milliliter centrifugation tube.
Re-suspend the isolated schizonts in complete RPMI media and increase the total volume to 40 milliliters. After centrifuging and discarding the supernatant, re-suspend the schizont in 10 milliliters of complete RPMI media. Then transfer one milliliter of this solution into a 1.5 milliliter micro centrifuge tube for each transfection and pellet the infected cells by centrifugation at 16, 000 G for five seconds.
Next, combine 100 microliters of the electroporation buffer at room temperature with five micrograms of target plasmid DNA in a 1.5 milliliter micro centrifuge tube. After centrifuging the infected cells, remove the supernatant and carefully re-suspend the cells in the prepared nucleoefector solution plus plasmid DNA. Then transfer the suspension into electroporation, cuvettes, and transfect using a suitable nucleofector program.
Next, add 100 microliters of complete RPMI media to the cuvette. Transfer the entire solution into a 1.5 milliliter micro centrifuge tube and place it on ice. Verify the parasitemia levels in the mice inoculated with the transfected schizont daily from two days post inoculation.
Once the infection is confirmed, start drug selection by administering the drug orally and drinking water offered ad libitum. Monitor parasitemia using blood smears starting from six days after the initiation of pyromethymine treatment. When parasitemia reaches at least 1%transfer the parasites to naive B6 mice to create blood stage parasite stocks.
Once parasitemia reaches 2%to 5%generate stocks and cryopreserve them. One day before mosquito infection, separate the female mosquitoes by placing a glove filled with water heated to 37 degrees Celsius on top of the cage containing both male and female mosquitoes. Use an insect aspirator to remove the female mosquitoes attracted to the heat source and transfer them to a smaller cage for infection.
On the day of mosquito feeding and infection, determine parasitemia in the infected B6 mice. Parasitemia between 2%to 5%is optimal for mosquito infection. After verifying the parasitemia, transfer the infection to the female mosquitoes by placing the anesthetized mice on the caged female mosquitoes under sternal recumbency.
Manually spread the front and back legs to provide the greatest surface area for mosquito feeding. Leave the infected mice in each cage for 15 minutes, checking every five minutes to ensure the mice are not awake. Once the feeding is complete and the mice are euthanized, dispose of them safely following the institution's guidelines.
To verify successful infection within the mosquitoes, isolate the mosquitoes midguts by removing the terminal abdominal segment with forceps 10 days post-infection. Then view the midguts under polarized light to detect the presence of oocysts. At 18 days post-infection, dissect five to 10 mosquitoes to evaluate the number of sporozoites present.
To isolate and count the sporozoites, carefully remove the head of the mosquito while applying pressure to the thorax with forceps or a fine dissecting needle. The salivary glands will remain attached to the removed head. Next, remove any additional mosquito debris from the salivary glands and place them in a micro centrifuge tube containing 400 microliters of mosquito dissection media.
Then disrupt the isolated salivary glands by passing them through a 30 gauge needle syringe 15 to 20 times, Afterward, place 10 microliters of the disrupted salivary glands in mosquito dissection media into a hemocytometer and count. Finally, re-suspend the salivary glands in the desired concentration of mosquito dissection media or PBS for injection into mice or long-term cryo-preservation. Like microscopy images depicting mature and immature P.berghei schizont in parasitized mouse blood cultures are presented.
Like microscopy images of a mosquito head with the salivary glands still intact during dissection, and the dissected salivary gland under lower and higher magnifications are shown. The generation of green fluorescence signal in the PB, GFP 11 infected HEPA GFP one to 10 host cells indicated the lysis of the parasitophorous vacuole. It's important to carefully remove the chaisson layer without disrupting the gradient interface.
Also, it takes a bit of practice to reliably remove the salivary glands along with the head during spores light isolation. Following isolation, transgenic sporozoites can be cryo-preserved or used fresh for in vivo or in vitro infection studies.
