Cloning of Plasmodium falciparum by single-cell sorting |
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Authors: | Jun Miao |
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Affiliation: | Department of Entomology, The Pennsylvania State University, University Park, PA 16802, USA |
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Abstract: | Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two-dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. |
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Keywords: | Plasmodium falciparum Transfection Parasite cloning Flow cytometry Cell sorting Red blood cells (RBCs) Green fluorescent protein (GFP) Forward angle light scatter (FSC) Side angle light scatter (SSC) |
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