Cloning of Plasmodium falciparum by single-cell sorting.
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.
Miao
Jun
J
Department of Entomology, The Pennsylvania State University, University Park, PA 16802, USA.
Li
Xiaolian
X
Cui
Liwang
L
D019976:Cloning, Organism / Q000379:methods*; D004912:Erythrocytes / Q000469:parasitology; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism; D006801:Humans; D049408:Luminescent Agents / Q000032:analysis / Q000378:metabolism; D010963:Plasmodium falciparum / Q000166:cytology* / Q000378:metabolism
D049408:Luminescent Agents; D049452:Green Fluorescent Proteins