Cloning of Plasmodium falciparum by single-cell sorting

  • Miao J
  • Li X
  • Cui L
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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. © 2010 Elsevier Inc.

Author-supplied keywords

  • Cell sorting
  • Flow cytometry
  • Forward angle light scatter (FSC)
  • Green fluorescent protein (GFP)
  • Parasite cloning
  • Plasmodium falciparum
  • Red blood cells (RBCs)
  • Side angle light scatter (SSC)
  • Transfection

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Authors

  • Jun Miao

  • Xiaolian Li

  • Liwang Cui

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