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Choukri B.M., I.Y. Gluzman, S. Goyard, S.M. Beverley, and D.E. Goldberg

A set of independent selectable markers for transfection of the human malaria parasite Plasmodium falciparum . Choukri B.M., I.Y. Gluzman, S. Goyard, S.M. Beverley, and D.E. Goldberg Presented by Roody Pierre-Charles. Important Terms. Selectable markers

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Choukri B.M., I.Y. Gluzman, S. Goyard, S.M. Beverley, and D.E. Goldberg

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  1. A set of independent selectable markers for transfection of the human malaria parasite Plasmodium falciparum Choukri B.M., I.Y. Gluzman, S. Goyard, S.M. Beverley, and D.E. Goldberg Presented by Roody Pierre-Charles

  2. Important Terms • Selectable markers • Done to verify that a chosen gene has been incorporated into the DNA of the organism to be modified. • They are predominantly antibiotic resistance gene markers • Transfection • The insertion of foreign genes into a nonbacterial cell • Assay • Test that measures the way in which an organism responds to a particular compound or substance

  3. The Mosquito, the primary carrier of Malaria • Malaria: transmitted by the bite of about 60 species of mosquitoes in the genus Anopheles

  4. Background • Malaria is the world's most important parasitic disease • Caused by intraerythrocytic protozoan parasites of the genus Plasmodium • Four species of Plasmodium are infectious to humans • Plasmodium falciparum • Plasmodium malariae • Plasmodium ovale • Plasmodium vivax • They annually cause clinical illness in 300 to 500 million people • 1.5 to 2.7 million deaths • mainly caused by P. falciparum

  5. Plasmodium falciparum • May occur in subtropical and tropical regions in almost all parts of the world • Characterized by : • Chills • Fever • Sweating • Organism often blocks the blood vessels of the brain • producing coma, delirium, and finally death

  6. Plasmodium falciparum • During the incubation period of malaria, the protozoan grows within cells in the liver • Few days before the first attack, the organisms invade the red blood cells • Destroy it in the course of their development, producing the typical febrile attack • Strains have shown resistance to chloroquine and other synthetic antimalarial drugs • ≈30 megabases in size • contains 14 chromosomes

  7. Plasmodium falciparum Infected red blood cell

  8. P. falciparum Life Cycle

  9. Sporozoites are transmitted into humans through the bite of an infected mosquito. • They travel to the liver, where they mature and divide into merozoites. • A single sporozoite can produce 5,000-10,000 merozoites.

  10. Merozoites infect red blood cells (RBCs). • They are generated either by sporozoites in the liver, or trophozoite division in RBCs.

  11. Trophozoites live within red blood cells (RBCs), feeding on hemoglobin. • They eventually undergo division into multiple merozoites, which invade more RBCs.

  12. Gametocytes are produced by differentiation of merozoites. • They are ingested by mosquitoes and mature into gametes in the mosquito’s gut. • Gametes will combine to form sporozoites, which can infect humans.

  13. A set of independent selectable markers for transfection of the human malaria parasite Plasmodium falciparum

  14. Rationale • To perform genetic manipulations to understand Plasmodium gene function, find additional selectable markers for the transfection of Plasmodium falciparum, and develop new methods of functional analysis of P. falciparum genome.

  15. Current Selectable markers for transfection of P. falciparum • Toxoplasma gondii dihydrofolate reductase genes • Confers resistance to methotrexate or pyrimethamine

  16. Drugs tested for Inhibiting Growthand Vectors Used • 10 different drugs were tested but two were selected: • Puromycin, nourseothricin,zeocin, phleomycin, phosphinothricin, blasticidin S, and G418 • Vectors chosen for this study were: • BSD (blasticidin S deaminase) • NEO (neomycin phosphotransferase II)

  17. Procedures • Strains • 3D7, HB3, Dd2, W2 • Cell Culture • Plasmid Constructs • Amplification of BSD • 5'-GGA AGA TGC ATG CCA AGC CTT TGT CTC AAG AAG AAT CCA CCC TC-3' • 5'-GAC GGG AAG CTTTGC TCC TCG GCC ACG AAG TGC-3' • Amplification of NEO • 5'-GGA AGATGC ATG GAT CGG CCA TTG AAC AAG-3' 5'-GAC GGG AAG CTTCTG TCT TTT TAT TGC CGA-3' • Drug Response Assays • P. falciparum Transfection and Selection of Transfectants • Southern Hybridization and Plasmid Recovery Analyses • Digested products were separated byelectrophoresis • transferred to Hybond N+ Nylon membrane • hybridized with 32P-labeled pBluescript II SK(+) probe • BSD Enzyme Assay • Neomycin Phosphotransferase Assay

  18. Vectors

  19. Why choose Blasticidin S and G418 for growth Inhibition? • Blasticidin S was chosen because it is potent in culture and its resistance determinantis encoded by a small ORF* • G418 was chosen, because it had already been used successfully as a selectable marker • Also called rifins, proteins derived from the antigenic-shift rif genes

  20. Range in Inhibition for blasticidin S and G418 blasticidinS from 0.15 to 0.45 µg/ml G418 from 150 to 380µg/ml

  21. BSD and NEO • After the virus was transfected, BSD culture was split into 3 lines BS1, BS2, BS5 • Parasites were detected in all lines after 4 wks • NEO culture also split into 3 lines but parasites were only detected in G3 and G5 after 4wks, not G10 even after 7wks. • 3D7 strain was transfected with pBluescript vector and used as the control

  22. To confirm that the transfected plasmids had been replicated by P. falciparum

  23. Monitoring of BSD and NEO Gene for Enzymatic Activity

  24. Conclusion • The two markers NEO and BSD were expressed in P. falciparum. • BSD is resistant for blasticidin S and NEO for G418. • A correlation appears to exist between the level of enzymaticactivity, the episome copy number, and the concentration of thedrug used. • The ability to use BSD and NEO markers for malaria transfection will set the stage for rescue of disrupted essential genes,for making gene libraries to complement naturally occurring orinduced mutants, or for other techniques that require the presenceof multiple independent markers. • The development of these resistancemarkers will allow better exploitation of the accumulating P.falciparum genomic information, lead to better understanding ofthe biology of the parasite, and facilitate development of drugand vaccinetargets.

  25. How does being a heterozygote for sickle-cell anemia block malaria? When oxygen levels drop in the bloodstreams of homozygotes, their red blood cells change from round to sickle-shaped. This shape causes them to get tangled up and clog the spleen and blood vessels. When the mosquito bites a human, it passes the malaria microbe into the human’s bloodstream. The microbe enters the blood cell, where it uses up oxygen which causes the cell to change to the sickle shape. Because of their shape,the sickle cells are more easily filtered out of the bloodstream by the spleen and are soon killed by white blood cells, which gather in the spleen. This keeps the infected cell from bursting open and infecting other cells! Answer by "Dr. Universe", http://www.wsu.edu/DrUniverse/evol2.html

  26. Questions?

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