Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 62 (1), 37-44

Two More Independent Selectable Markers for Stable Transfection of Leishmania

Affiliations

Two More Independent Selectable Markers for Stable Transfection of Leishmania

D J Freedman et al. Mol Biochem Parasitol.

Abstract

Genetic transformation of Leishmania has relied upon two exogenous selectable markers, neo and hyg, encoding resistance to G418 and hygromycin B respectively. There is a need for multiple independent selectable markers, since Leishmania is diploid and experimental sexual crosses are not currently feasible. Here we report on the development of two additional markers: pac, conferring resistance to the glycopeptide antibiotic puromycin, and phleo, conferring resistance to the DNA-binding drug phleomycin. We constructed a set of four analogous shuttle vectors with these four markers, using DNA segments flanking the Leishmania major H region hmtxr gene to provide information required for expression. These constructs (pHM-NEO, pHM-HYG, pHM-PAC and pHM-PHLEO) were successfully transfected into L. major, mostly with efficiencies comparable to those observed with previous DHFR-TS-based neo and hyg-containing constructs. The exception was pHM-PHLEO, which transfected 30-fold less efficiently; this may be related to the nonenzymatic mechanism of resistance encoded by phleo. All four constructs were shown to replicate extra-chromosomally. Stable transfectants bearing all paired combinations of pHM constructs were obtained by a second round of transfection. These data show that the four markers are functionally independent and in conjunction with the Leishmania N-acetylglucosaminyl transferase gene, brings the number of selectable markers available in Leishmania to five.

Similar articles

See all similar articles

Cited by 26 PubMed Central articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback