A homologous recombination strategy to analyze the vinblastine resistance property of the V-circle in Leishmania

Abstract

The generation of extrachromosomal DNA elements in Leishmania sp. can occur naturally or during in vitro selection with drugs. Previously, we had established a strong association between V-circle amplification and drug resistance in L. enriettii stepwise selected with increasing concentrations of vinblastine. Further, we demonstrated the presence of the lemdr1 gene in the amplified V-circle and subsequent functional analysis by transfection of this gene alone confirmed its role in conferring a drug-resistant phenotype, but the level of resistance was significantly lower than in cell lines obtained from stepwise drug selection. The aim of this work was to determine if other genes either on the V-circle or elsewhere in the genome were necessary for expression of vinblastine resistance. We report here the development of a homologous recombination method to convert the entire V-circle from the LeV160 cell line into a shuttle vector and further the targeted disruption of specific sites within the V-circle. Our results clearly demonstrate that the V-circle alone is sufficient to confer full vinblastine resistance and the disruption of the lemdr1 locus destroys the ability of the V-circle to confer vinblastine resistance.