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Please use this identifier to cite or link to this item: http://hdl.handle.net/10204/4104

Title: Establishing malaria parasite transfection technology in South Africa
Authors: Van Brummelen, AC
Becker, JVW
Mancama, DT
Hoppe, HC
Keywords: Plasmodium falciparum
Anti-malarial drug
Anti-malarial drug resistance
Transfection
Green fluorescent protein
GFP
Luciferase
Malaria parasite
Biochemistry
Molecular biology
Issue Date: Jan-2010
Citation: Van Brummelen, AC,Becker, JVW,Mancama, DT and Hoppe, HC. 2010. Establishing malaria parasite transfection technology in South Africa. 22nd South African Society for Biochemistry and Molecular Biology Congress. Bloemfontein, South Africa, 18-20 January 2010, pp 2
Abstract: The most important contributing factor to the current malaria crisis is the rapid spread of parasite resistance to available anti-malarial drugs. Anti-malarial drug resistance is critical and the need for compounds with novel modes of action is beyond the point of urgency. A rational and a deliberate target-based drug discovery strategy as opposed to a “shotgun” screening-approach requires specific, well-characterized and validated drug targets. Gene manipulation is the only means by which the functions of individual proteins in living cells can confidently be confirmed and is thus a fundamental requirement in target-based drug discovery. Genetic manipulation of the malaria parasite, Plasmodium falciparum, is usually performed via plasmid-based transfection similar to those used in other eukaryotic organisms. However, a major obstacle faced by malaria research laboratories world-wide is the poor transfection efficiencies of current protocols due to the unique technical problems faced i.e. AT-richness and intracellular location of the organism. As a result such successful transfection often requires prolonged periods (up to 2-3 months) of constant and patient culturing and selection. In addition, plasmids usually have a complicated composition and require lengthy cloning manipulations to prepare. Improvements to the methodology would have wide impact and is constantly being sought. In order to establish malaria parasite transfection technology in South Africa, firefly luciferase and green fluorescent protein (GFP) reporter constructs were prepared. In attempt to simplify these constructs, a var intron (PFC0005w), previously reported to have bidirectional promoter activity was utilized to drive expression through two genes (i.e. the antibiotic-resistance gene, human dhfr and the reporter gene) in a head-to-head orientation. In addition, protocols were adjusted by including DNA packaging reagents to improve uptake into the parasite and by using shaking instead of stationary parasite cultures to improve the parasite proliferation and selection rate. Successfully transfected parasites were selected by the antifolate WR99210. The first transfected parasites appeared after 33 days indicating that the dhfr gene was expressed at sufficient levels to produce drug resistant parasites. Luciferase and GFP expression was also confirmed. The established technology will be applied in the future to validate potential targets as identified by CSIR functional genomics investigations.
Description: 22nd South African Society for Biochemistry and Molecular Biology Congress. Bloemfontein, South Africa, 18-20 January 2010
URI: http://hdl.handle.net/10204/4104
Appears in Collections:Systems biology
General science, engineering & technology

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