1National Institute of Allergy and Infectious Diseases, National Institutes of Health, 2School of Life Science, Xiamen University
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Pattaradilokrat, S., Li, J., Su, X. Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites. J. Vis. Exp. (47), e2365, doi:10.3791/2365 (2011).
Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.
Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.
Aseptic techniques must be applied to all materials that will be administrated into animals to avoid inadvertent introduction of exogenous infectious agents into mice that can confound experimental outcomes.
1. Infection of Laboratory Mice with Blood-stage Malaria Parasites
2. Mixed Clone Infection of Mice
3. Feeding Mosquitoes
4. Dissecting Mosquito Midguts and Counting Oocysts
5. Collecting Sporozoites from Mosquitoes by Centrifugation
6. Cloning Progeny using Limited Dilution
7. Characterization of Progeny using Genetic Markers
8. Cryopreservation of Blood-stage Malaria Parasites
9. Representative Results:
In a successful experiment, 5-10% of the cloned lines will be independent recombinant progeny.
Figure 1. A schematic representation of the life cycle and genetic recombination events during a genetic cross. Genetic recombination events take place during the early phase of development in the mosquito. Following the fertilization of "haploid" male and female gametes of different genetic backgrounds in the midgut of the mosquito, "diploid" zygotes are formed. The zygote constitutes the only diploid stage of the parasite life cycle; meiosis follows within 12 hrs of fertilization, and all subsequent stages in the mosquito and mammals remain haploid. The resulting zygotes develop into ookinetes and oocysts. Growth and mitotic division of each oocyst produces thousands of sporozoites, which are released into mosquitoes' hemoceal. Those sporozoites that migrate to salivary glands are in position to be transmitted to a mammalian host, where development of the liver and red blood cell stages takes place. During the course of the red blood cell cycles, some parasites can differentiate into mature male and female gametocytes. The parasite life cycle is completed when these gametocytes are taken up by another mosquito, perpetuating transmission.
Figure 2. Inheritance of nuclear genes in malaria parasites and production of recombinant progeny. Genetic recombination can be generated through chromosomal reassortment or by crossover events. Homozygous progeny produced by selfing between gametes of the same parental clone 1 or 2 (A and D), respectively. B) Heterozygous progeny produced by cross-fertilization between gametes of the two different parental clones, recombinant nuclei (blue ovals) being formed by chromosomal reassortment alone. C) Heterozygous progeny produced from cross-fertilization between gametes of the two different parent clones, recombinant nuclei (red ovals) being formed by crossovers between the non-sister chromatids of the homologous chromosomes.
Figure 3. Morphology of the rodent malaria parasite Plasmodium yoelii. A) ring stage, B) trophozoite, C) schizont, D) merozoites, E) immature male gametocyte, F) mature male gametocyte, G) immature female gametocyte, H) mature female gametocyte, I) midgut with oocysts on day 10 post feeding (mature oocysts indicated by arrows; 200x magnification), J) mosquito salivary glands (100x magnification), K) sporozoites (400x magnification).
We demonstrate the techniques for the production of a genetic cross in the rodent malaria Plasmodium yoelii, which is also applicable to production of genetic crosses in other rodent malarias. Infections of mice with single parental clones are usually performed to determine successful transmission of the parental parasites to ensure that the parents are competent in producing functional gametes before performing a cross.
Successful transmission through a mosquito is influenced by multiple factors including temperature of the insectary, species of rodent malaria parasites used, and the doses of blood-stage parasite (inoculum size). While transmission of P. berghei is normally achieved at 19-21°C 13, transmission of P. yoelii and P. chabaudi is routinely performed at 23-25°C 14. Ookinetes of P. berghei fail to develop into oocysts at temperatures lower than 16°C or higher than 24°C 15. In addition to temperature, inoculum size can influence multiplication rate of malaria parasites in the blood. Although injections of a large inoculum size (5 x 106 iRBC or more) will yield higher parasitemia at very early stages of infection, it does not necessarily lead to larger numbers of gametocytes or higher infectivity to mosquitoes. Gadsby and others (2009) 16 showed that gametocytes of P. chabaudi adami are present in the blood from day 3 to day 20 (peak at day 13) post infection of 1 x 106 iRBC; however, day 6 post infection is the only day upon which mosquitoes become infected. Also, administration of a large inoculum size of fast-growing and virulent parasite strains such as clones N67 (nigeriensis), 17XL, and YM of P. yoelii, clone ANKA of P. berghei, and clone DS of P. chabaudi adami often results in severe anemia and pathology in mice, which can cause a significant drop of mouse body temperature. As a result, mice become less infective to mosquitoes (unpublished observations). Nevertheless, transmission of rodent malaria parasites has been conducted with a standard inoculum size (106 iRBC). It has been found that P. berghei and P. yoelii are transmissible to mosquitoes from days 3 to 5 after blood stage-induced infection in mice17, whereas P. chabaudi chabaudi and P. chabaudi adami are only transmissible to mosquitoes on day 6 after blood stage-induced infections in mice16, 18.
Factors that influence production of recombinant progeny include the proportions of gametocytes of the two parental strains in mice. In theory, the maximum production of recombinant progeny occurs when gametocytes of both sexes of the parental clones are in equal number and self-fertilization and cross-fertilization occur at the same frequency. Under this condition, half of the resulting zygotes will be hybrids and the remainder will consist of equal proportions of the two parental clone lines. Production of recombinant progeny clones will be greatly reduced when the proportion of gametocytes is biased toward one parental clone, leading to disproportional self-fertilization. Moreover, parasites often have different growth rates and may have different capability in producing gametocytes16, 19. Therefore, it is necessary to optimize the proportions of parental parasites in the mixture to be injected into the mice for mosquito feeding.
The time to clone parasites from blood after mosquito feeding is also an important factor to consider. It is preferable to clone the progeny of the genetic cross when parasitemia is between 0.1-1.0% (before too many cycles of replication in the blood) to avoid cloning duplicated clones. Fast- or slow-growing parasites may come out in large numbers at certain periods of time, and cloning at the peaks of fast- or slow-growing parasites will end up with clones having the same phenotypes.
In conclusion, performing genetic crosses using rodent malaria parasites is relative easy and much cheaper than performing a genetic cross of the human malaria parasite P. falciparum, which requires infection of a nonhuman primate. Recombinant progeny of the genetic crosses are useful tools for construction of genetic linkage maps and for mapping of important malarial traits including parasite development, drug resistance, and virulence.
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We thank Drs Randy Elkins, Robin Kastenmayer, Ted Torrey, Dan Pare and Tovi Lehman for critical reading of manuscripts. This work was supported by the Intramural Research Program of the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, and by the 973 National Basic Research Program of China, #2007CB513103. We thank NIAID intramural editor Brenda Rae Marshall for assistance.
|Glyerolyte 57 solution||Cenmed||4A7833|
|Mouse Mus musculus||Charles River Laboratories||Female, inbred, strain Balb/C|
|Heat-inactivated calf serum||Invitrogen||26010-066|
|Phosphate buffered saline (PBS) solution||Invitrogen||10010-072||pH 7.4; Cell Culture grade|
|Malaria parasite Plasmodium y–lii y–lii 17XNL(1.1)||MR4||MRA-593||deposited by DJ Carucci|
|Malaria parasite Plasmodium y–lii nigeriensis N67||MR4||MRA-427||deposited by W Peters, BL Robinson, R Killick Kendrick|
|Mosquito Anopheles stephensi||MR4||MRA-128||deposited by MQ Benedict|
|Cellometer automatic cell counter||Nexcelom Bioscience||Cellometer Auto T4|
|Cellometer CP2 disposable hemacytometer||Nexcelom Bioscience||Cellometer CP2|
|High Pure PCR template preparation kit||Roche Group||11 796 828 001|
|Calcium chloride||Sigma-Aldrich||C5670||Cell culture tested; insect cell culture tested|
|Giemsa stain, modified||Sigma-Aldrich||GS500|
|Ketamine hydrochloride||Fort Dodge Animal Health||NDC 0856-2013-01||Pharmaceutical grade; concentration to 100 mg/mL|
|Potassium chloride||Sigma-Aldrich||P5405||Cell culture tested; insect cell culture tested|
|Sodium chloride||Sigma-Aldrich||S5886||Cell culture tested; insect cell culture tested|
|Trisodium citrate dihydrate||Sigma-Aldrich||S4641|
|Xylazine||Akorn Inc||4811-20ml||Pharmaceutical grade; concentration to 20 mg/mL|
|Glass wool||VWR international||32848-003|
|Glass capillary (1 μL)||VWR international||53440-001|
|Hemocytometer||VWR international||15170-168||Complete chamber set|
|Homogenizer||VWR international||KT749520-0090||Pestle with matching tube, 1.5 mL|
Maintenance of laboratory mice
Females of inbred laboratory mouse strain BALB/c, aged 5 to 8 weeks old, are used in the study. Mice are housed in a standard solid-bottom polycarbonate cage with wire-bar lid, equipped with feeder and a water bottle. Mice are maintained at a constant temperature (25 ± 1°C) on 12:12 hour light:dark cycle. Mice are allowed to feed on 2018S Harlan Teklad Global 19% protein extruded rodent diet (sterilizable; from Harlan-Teklad) and supplied with acidified drinking water ad libitum. Experiments on animals are performed in accordance with the guidelines and regulations set forth by the Animal Care and Use Committee at the National Institute of Allergy and Infectious Disease under protocol LMVR11E (National Institutes of Health, Bethesda, Maryland).
Maintenance of laboratory mosquit–s
Mosquit–s are from a laboratory-bred colony of Anopheles stephensi. The adults are maintained in nylon cages kept in a temperature- and humidity-controlled room (23 to 25°C for Plasmodium y–lii and Plasmodium chabaudi, and 19 to 21°C for Plasmodium berghei; 80 to 95% humidity; on 12:12 hours light:dark cycle). Adult mosquit–s are fed with 10% glucose and 2.00% para-aminobenzoic acid (PABA) supplemented water solution. To obtain high-quality adults, 500 larvae are grown in a low-density condition in 1 L of distilled water in a 1,000-cm3 open dish supplied with approximately 1 mg of sodium bicarbonate. After hatching, the larvae are given tetramin powder (PETCO) until they develop into the pupa stage and are transferred to the adult mosquito cages for emerging.
Microscopic examination of thin blood smears stained with Giemsa stain
Using clean scissors snip off the tip (1.0 mm) of the infected mouse’s tail. Place one drop (0.5-1.0 μL) of tail blood onto a clean specimen slide. Mouse will stop bleeding in 1-2 min. Place a clean spreader slide on top of the blood drop, maintaining it at a 45° angle relative to the specimen slide, and allow the blood to adsorb to the entire width of the spreader. Hold the specimen slide and push forward the spreader slide rapidly and smoothly to produce a thin smear. Let the blood film dry, and then immerse the slides in absolute methanol. Allow the slide to air dry once more before covering it with Giemsa stain (10% Giemsa dye in distilled water). After incubating the thin blood films for 10-15 min at room temperature, carefully rinse the slides with tap water and let it air dry. Examine the number of infected red blood cells (iRBC; see Figure 3 for morphology of infected RBC) under a light microscope with immersion oil at 1000x magnification (with 100x objective lens) and calculate parasitemia (the number of iRBC per 100 RBC counted). Different strains of malaria parasites vary in growth rate and pathogenicity. Monitoring of blood stage parasitaemias can be performed 24hrs after injections, depending on the dose of the blood stage malaria parasites. For example, mice will be microscopically positive 24 hrs when injected with 107 infected RBC intraperitoneally or 106 infected RBC intravenously.
Measurement of red blood cell density
Like the levels of parasitaemias, red blood cell (RBC) density in infected mice varies throughout the course of infection. RBC density should be measured within 1-2 hrs before the start of the single- and mixed-clone infection and the cloning experiments. There are two methods for measurement of RBC density: a manual counting using Neubauer hemocytometer and an automatic counting using a Cellometer (Nexcelom Bioscience). In both methods, withdraw 1 μL of mouse tail blood using a glass capillary (VWR) and dilute in 10 mL of PBS and mix well. To use a Neubauer hemocytometer, load 20 μL of the suspension onto the hemacytometer. Place the hemacytometer on a light microscope with 10x objective lens. The hemacytometer contains a grid divided into 9 large squares, and 4 large squares at the corner are further divided into 16 small squares. Count the total number of cells in each of the 16 small squares in the four corner squares. To avoid counting bias or counting cells that overlap a grid line, count a cell as "in" if it overlaps the top or right lines and "out" if it overlaps the bottom or left lines. Estimate the number of cells per one small square and divide by 0.00625 (the volume of one small square is 6.25 nL). This yields the number of cells per microliter (μL). From this data, calculate the final red blood cell density by multiplying with 10,000 (a dilution factor). Rinse the cover slip and counting chamber with distilled water and 70% ethanol; air dry. Alternatively, load 20 μL of the suspension onto a Cellometer counting chamber slide. Insert the slide into a Cellometer slide chamber (the reader). Start the Cellometer software, select the "red blood cell" option, and enter a dilution factor of 10,000. Record the RBC density.