Generating Genetically Modified Plasmodium berghei Sporozoites

All research involving vertebrate animals in our laboratory was performed in compliance with the University of Georgia animal use guidelines and protocols.

1. Generation of P. berghei -infected mice

1. Initiate blood-stage infection in male or female, 6-8-week-old C57BL/6 (B6) mice using wild-type P. berghei parasites. To do this, transfer cryopreserved P. berghei-infected blood (2 x 10⁵ infected RBCs), reconstituted in 400 µL of phosphate-buffered saline (PBS), into one or two donor mice intraperitoneally (i.p.).
2. Transfer fresh blood collected through retro-orbital bleeding from the donor mice at 5 days post-infection (d.p.i.) into two naïve B6 mice. To do this, collect 200 µL of blood, reconstitute with 300 µL of PBS, and inject 200 µL i.p. into the recipient mice. Ensure that the parasitemia in the donors at this time point is 2%-5%^19,20.
3. Monitor parasitemia as previously described^19,20, starting from 2 d.p.i. in the recipients. When parasitemia ranges from 3%-5% (around 5 d.p.i.), euthanize the recipient mice and then collect blood through cardiac puncture or retro-orbital bleeding. Euthanize the mice using carbon dioxide inhalation followed by cervical dislocation, or according to institutional guidelines.
   ​NOTE: Once parasitemia exceeds 5%, the transfection efficiency may be reduced due to the increased prevalence of multiply infected RBCs.

2. Generation of schizonts in culture

1. Collect blood from the infected mice in step 1.3 (approximately 2 mL in total from two mice) into 5 mL of Alsever's solution (see Table of Materials) in a sterile environment. Perform steps 2.1 to 3.12 under sterile conditions.
   NOTE: Sterile conditions include wearing gloves, wiping gloves and surfaces with 70% ethanol, employing sterile consumables and materials, and working within a biosafety cabinet.
2. Centrifuge the blood for 8 min at 450 x g at room temperature (RT). Discard the supernatant and resuspend the cell pellet in 10 mL of complete RPMI media (RPMI 1640 with 20% fetal bovine serum [FBS] and 1% penicillin-streptomycin, v/v).
3. Centrifuge for 8 min at 450 x g at RT. Resuspend the pellet in 25 mL of complete RPMI media and transfer to a T75 culture flask with a filter cap.
4. Place in an incubator at 36.5 °C and 5% CO₂, while gently shaking to keep the cells in suspension for 16 h.
5. At the 16 h time point, check for schizont development. To do this, collect 500 µL of the sample, centrifuge at 450 x g for 8 min, resuspend the pellet in 10 µL of complete RPMI media, and prepare a thin blood smear. Stain the blood smear with Giemsa stain¹⁹ (see Table of Materials) and determine the frequency of schizonts (Figure 1).
6. For optimal transfection efficiency, 60%-70% of the parasites should be in the schizont stage. If fewer than this threshold is determined, continue culturing as in step 2.4 and check every hour to ensure the above threshold is crossed before proceeding.

3. Transfection of Plasmodium schizonts

1. Prepare a gradient centrifugation buffer by reconstituting 13 mL of density gradient stock medium (see Table of Materials) with 1.44 mL of 1.5 M NaCl and 5.6 mL of 0.15 M NaCl in a 50 mL centrifugation tube.
2. Transfer 25 mL of blood culture to a fresh 50 mL centrifugation tube. Carefully layer 20 mL of gradient centrifugation buffer to the bottom of the centrifugation tube using a narrow glass pipette to create a gradient column. Take care not to disturb the liquid layers and interfaces and ensure a clear division between the buffer and the media.
3. Centrifuge for 20 min at 450 x g with no brake at RT. Using a Pasteur pipette, carefully remove the schizont layer¹⁵ located at the interface of the culture media and the gradient centrifugation buffer, and place it in a new 50 mL centrifugation tube.
4. Resuspend the isolated schizonts in complete RPMI media and bring up the total volume to 40 mL. Centrifuge at 450 x g for 8 min at RT and discard the supernatant.
5. Resuspend the schizonts in 10 mL of complete RPMI media. Then, transfer 1 mL of this solution into a 1.5 mL microcentrifuge tube for each transfection, and centrifuge at 16,000 x g for 5 s to pellet the infected RBCs. The infected RBCs derived from two mice in the above manner can be used for up to 10 separate transfections.
6. Combine 100 µL of the electroporation buffer (nucleofector solution from the electroporation kit; see Table of Materials) at RT with 5 µg of circular or linearized target plasmid DNA (in up to a maximum of 10 µL volume), as applicable for each transfection in separate 1.5 mL microcentrifuge tubes. A "no plasmid" sample must be included as a control for transfection and antibiotic selection.
7. Remove the supernatant after centrifugation from step 3.5 and carefully resuspend the infected RBCs in the prepared nucleofector solution + plasmid DNA (from step 3.6).
8. Transfer the suspension (nucleofector solution + plasmid + schizonts; 110 µL maximum) into electroporation cuvettes. Transfect using a suitable nucleofector program (U-033, see Table of Materials).
   NOTE: Ensure that steps 3.8 to 3.10 are performed at RT.
9. Add 100 µL of complete RPMI media to the cuvette and transfer the entire solution (now 200 µL total volume) into a 1.5 mL microcentrifuge tube and keep at RT.
10. Inject the 200 µL solution of transfected parasites into mice intravenously (i.v.), working to minimize the time between transfection and the final injection into mice.
    ​NOTE: Transfected schizonts are injected into mice less than 5 min after electroporation to maximize the efficiency of the protocol.

4. Selection of the transfected parasites

1. Check the parasitemia levels in the mice inoculated with the transfected schizonts daily from 2 d.p.i. to verify infection.
2. Once the infection is verified, start drug selection using oral administration of the drug in drinking water, offered ad libitum.
   NOTE: Pyrimethamine was employed as the selectable drug marker for the plasmid. In this case, 17.5 mg of pyrimethamine was added to 250 mL of clean water (final concentration of 70 mg/L). In addition, 10 g of sugar was added to this water to encourage adequate consumption of the pyrimethamine-containing water by mice.
3. Monitor parasitemia every two days using blood smears made with 5 µl of blood, starting from 6 days after the initiation of pyrimethamine treatment. The lack of detectable parasites after 15 days indicates a failed transfection; in this case, reinitiate the process from step 1.1 for a fresh attempt.
4. When parasitemia reaches at least 1%, transfer the parasites to naïve B6 mice for the creation of blood-stage parasite stocks. Continue selection in the newly infected mice until parasitemia reaches 2%-5%, at which point generate stocks and cryopreserve them²¹.

5. Infection of mosquitoes with the transgenic lines

1. Infect B6 mice with 200 µL of blood containing the selected parasites (2 x 10⁵ infected RBCs/mouse, i.v.). Determine parasitemia in these mice on the day of mosquito feeding and infection. Parasitemia between 2%-5% is optimal for the infection of mosquitoes. Once verified, immediately transfer the infection to the female Anopheles stephensi mosquitoes according to steps 5.2-5.4).
   NOTE: Incubators containing mosquitoes for infection should be maintained at 20.5 °C, at 80% humidity, and on a 12 h light/dark cycle (lights on between 07:00 a.m. to 07:00 p.m.).
2. The day before mosquito infection, separate the female mosquitoes by placing a heat source on top of the cage containing both male and female mosquitoes, such as a thin plastic bladder or glove filled with water heated to 37 °C. Remove the female mosquitoes, which are attracted to the heat source, using an insect aspirator and transfer these to a separate, smaller cage for infection.
   NOTE: Male mosquitoes can be distinguished from female mosquitoes by their slightly reduced body size and plumose antennae.
3. Inject the infected mice with a general anesthetic (e.g., 2% tribromoethanol/avertin). Ensure complete anesthesia by firmly pinching the foot pad of each mouse. If they do not react, they are sufficiently sedated and ready to transfer the infection into mosquitoes.
4. Place the anesthetized mice on top of the caged female mosquitoes (from step 5.2) under sternal recumbency. Manually spread the front and back legs to provide the greatest surface area for mosquito feeding. Leave the infected mice over each cage for a total of 15 min, checking every 5 min to ensure the mice are not awake. Once feeding is complete, euthanize the mice using cervical dislocation, or as per the institutional animal use protocol, and dispose of them safely.

6. Collection of sporozoites

1. Check mosquito midguts for oocysts at approximately 10 d.p.i. to verify successful infection and the development of Plasmodium within the mosquitoes. Isolate mosquito midguts from the abdomen by removing the terminal abdominal segment using forceps. Visualize the midguts under polarized light for the presence of oocysts²².
2. Wild-type sporozoites are expected to be present in the salivary glands of mosquitoes 18-21 days after feeding on infected mice. At day 18, dissect 5-10 mosquitoes to assess sporozoite numbers.
   NOTE: Mosquitos are washed and killed in ethanol. Subsequently, dead mosquitos are washed twice with PBS to remove debris prior to dissection.
3. To isolate and count the sporozoites, carefully remove the head of the mosquito while applying pressure to the mosquito thorax using forceps or a fine dissecting needle. The salivary glands remain attached to the removed head (Figure 2).
4. Remove any additional mosquito debris from the salivary glands and place in a microcentrifuge tube containing 400 µL of mosquito dissection media (Dulbecco's modified Ealge medium [DMEM] with 1% mouse serum, 100 U/mL penicillin, and 100 µg/mL streptomycin; see Table of Materials).
5. Disrupt the isolated salivary glands by passing through a 30 G needled syringe 15-20 times. Place 10 µL of the disrupted salivary glands in mosquito dissection media into a hemocytometer and count. Resuspend in the desired concentration of mosquito dissection media or PBS for injection into mice, inoculation into cell cultures, or long-term cryopreservation^23,24.