CharacterizationoftheputativesurfaceproteinTLPin.pptVIP

41% of the World Population live in endemic areas Estimated 700,000 to 2.7 million malaria deaths annually, 75% of them African children TLP (Thrombospondin Like Protein) discovered via micro array (Hayward et al., 2000) Putative surface protein? Shares conserved domains with: Experimental Plan: 1. Determine stage TLP expressed in Wild Type - Quantitative Real-Time PCR 2. Verify TLP knockout gene disruption - Diagnostic PCR - Southern Blot * * Characterization of the putative surface protein TLP in Plasmodium berghei and Plasmodium falciparum Ryan M Harrison1,2, Cristina K Moreria3, Catherine Lavazec3, Thomas J Templeton3 1 Gateways to the Laboratory Program, Weill Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD-PhD Program, New York, NY 2 Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 3 Department of Immunology and Microbiology, Weill Cornell Medical College, New York, NY Malaria in Context /malaria/facts.htm Plasmodium Life Cycle Sporozoite Merozoite Asexual/ Gametocyte Zygote Ookinete Oocyst Plasmodium, Encyclopedia of Life Sciences, 2006 History of TLP ? ? - - - - - - Asexual Stages ? ? + + - - - - Ookinete ? ? - - - + - + Hemocoel Sporozoite ? ? - - + + - + Salivary Gland Sporozoite cytoplasm membrane cytoplasm membrane cytoplasm membrane cytoplasm membrane ? TLP CTRP TRAP CSP ? Project Goals Hypothesis: The TLP protein is predominately expressed in the sporozoite life cycle stage of both P. falciparum and P. berghei P. berghei TLP Expression P. falciparum TLP Expression Verification of gene disruption A B C D WT TLP P. berghei A B C D P. falciparum A B C D A B C D WT TLP A B C D Diagnostic PCR Integrated cassette gDNA gDNA Verification of gene disruption Southern blot design gDNA gDNA NcoI HindIII Hybridization Probe Wildtype TLP knockout 7,600bp gDNA gDNA NcoI HindIII Hybridization Probe 9,700bp HindIII HindIII 2000bp Integrated Cassette Verification of gene disruption 1,65