Blasticidin resistance: a new independent marker for stable transfection of Leishmania.

Protozoan parasites of the genus Leishmania are the etiologic agents of a spectrum of human diseases referred to as leishmaniasis. Leishmania alternates life stage between an intracellular amastigote form residing within the phagolysosome of vertebrate macrophage and an extracellular promastigote form living in the gut of sand flies. Molecular characterization of the genome of Leishmania has been greatly facilitated by the development of stable transfection methods, expression vectors and gene knockouts [1,2]. Stable transfection of Leishmania and trypanosomes requires the use of dominant selectable markers, and a number of independent markers have been used successfully. These include neomycin phosphotransferase (NEO) [3]; hygromycin phosphotransferase (HYG) [4], streptothricin acetyltransferase (SAT) [5], puromycin acetyltransferase (PAC) [6] and the bleomycin binding protein from Streptoalloteichus hindustanus (PHLEO) [6], which confer resistance to G418, hygromycin B, nourseothricin, puromycin and phleomycin respectively. In addition, overexpression of N-acetylglucosamine-1-phosphate transferase (NAGT) or dihydrofolate reductasethymidylate synthase (DHFR-TS) can confer resistance to tunicamycin and methotrexate respectively, allowing their use as selectable markers on episomal vectors [7,8]. Since Leishmania is diploid and lacks an experimentally manipulable sexual cycle, generation of null mutants requires the inactivation of two alleles [4]. Typically this is accomplished by two rounds of targeting with independent selectable markers, with a third marker needed to generate an ‘add-back’ construct restoring gene expression; this is an essential control for biological function [9]. While the use of loss of heterozygosity approaches can reduce the number of markers needed for null mutant construction [10], with the current repertoire of markers one can manipulate Abbre6iations: BSD, gene encoding blasticidin deaminase. * Corresponding author. Tel.: +1-314-7472630; fax: +1314-7472634. E-mail address: [email protected] (S.M. Beverley)