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

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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

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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

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

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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

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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

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Plasmodium falciparum
Infected red blood cell
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P. falciparum Life Cycle
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• 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.

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

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

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

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

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Current Selectable markers for transfection of P.
falciparum

• Toxoplasma gondii dihydrofolate reductase genes
• Confers resistance to methotrexate or
  pyrimethamine

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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)

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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 CTT TGC TCC TCG GCC ACG AAG
  TGC-3'
• Amplification of NEO
• 5'-GGA AGA TGC ATG GAT CGG CCA TTG AAC AAG-3'
• 5'-GAC GGG AAG CTT CTG 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 by
  electrophoresis
• transferred to Hybond N Nylon membrane
• hybridized with 32P-labeled pBluescript II SK()
  probe
• BSD Enzyme Assay
• Neomycin Phosphotransferase Assay

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Vectors
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Why choose Blasticidin S and G418 for growth
Inhibition?

• Blasticidin S was chosen because it is potent in
  culture and its resistance determinant is 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

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

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To confirm that the transfected plasmids had
been replicated by P. falciparum
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Monitoring of BSD and NEO Gene for Enzymatic
Activity
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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 enzymatic activity, the episome copy number,
  and the concentration of the drug 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
  or induced mutants, or for other techniques that
  require the presence of multiple independent
  markers.
• The development of these resistance markers will
  allow better exploitation of the accumulating
  P. falciparum genomic information, lead to better
  understanding of the biology of the parasite, and
  facilitate development of drug and vaccine
  targets.

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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 humans 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

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Questions?