RESUMEN
Experimental reverse genetic approaches have proven powerful in the study of the biology of the malaria parasite. The murine malaria model parasite Plasmodium berghei is the genetically most amendable Plasmodium species and allows full access to the entire life cycle in vivo. Here, we describe a next-generation, highly versatile transfection vector set that facilitates advancing experimental genetic strategies towards a genome-wide scale. Through 36 consecutive cloning and 17 subcloning steps an optimized vector set was generated from the standard transfection plasmid. These targeting vectors, collectively referred to as the Berghei Adaptable Transfection (pBAT) plasmids, contain key elements that permit recycling of the drug-selectable cassette, robust green fluorescent labelling of recombinant parasites, carboxy-terminal tagging of target proteins with a red fluorescent-epitope tag fusion, and expression of heterologous genes. The vectors were further optimized for small size, versatile restriction endonuclease recognition sites and potential exchange of individual vector elements. We show that stable integration into a transgene expression site, an intergenic locus at a synteny breakpoint on P. berghei chromosome 6, is phenotypically silent and generated a bright green fluorescent parasite line for imaging applications. We provide an example, P. berghei actin 2, for targeted gene deletion and illustrate that the positive selection marker can be recycled, thereby permitting multiple rounds of genetic manipulations. We propose that the vectors described herein will greatly facilitate functional assignment to predicted and orphan Plasmodium gene models by multiple experimental genetics approaches.
