Improvement of an in vitro drug selection method for generating transgenic Plasmodium berghei parasites

Background: Reverse genetics approaches have become powerful tools to dissect the biology of malaria parasites. In a previous study, development of an in vitro drug selection method for generating transgenic parasite of Plasmodium berghei was reported. Using this method, two novel and independent selection markers using the P. berghei heat shock protein 70 promoter was previously established. While the approach permits the easy and flexible genetic manipulation of P. berghei, shortcomings include a low variety in promoter options to drive marker gene expression and increased complexity of the selection procedure. In this study, addressing these issues was attempted. Methods: To secure a variety of promoters, the use of a P. berghei elongation factor-1α promoter for marker gene expression was attempted. To simplify the procedure of in vitro selection, the establishment of a two cell-cycle culture method and its application for drug selection were attempted. Results: The P. berghei elongation factor-1α (pbef-1α) promoter, which is commonly used to drive marker gene expression, was successfully applied as an alternative promoter model for marker gene expression, using the parasite's codon-optimized marker sequence. To simplify the in vitro selection method, a two cell-cycle culture method in which the merozoite was released by filtration of the culture containing matured schizont-infected erythrocytes was also developed and successfully applied for drug selection. Conclusion: The pbef-1α promoter was successfully applied in an in vitro selection system. The in vitro selection procedure also could be simplified for practical use using a two cell-cycle culture method. These improvements provide a more versatile platform for the genetic manipulation of P. berghei.