Two more independent selectable markers for stable transfection of Leishmania.
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.
Freedman
D J
DJ
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.
Beverley
S M
SM
D000818:Animals; D001483:Base Sequence; D016054:DNA, Protozoan / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D017125:Genes, Protozoan; D005819:Genetic Markers; D005822:Genetic Vectors; D005839:Gentamicins / Q000494:pharmacology; D006921:Hygromycin B / Q000494:pharmacology; D018320:Leishmania major / Q000187:drug effects / Q000235:genetics*; D008969:Molecular Sequence Data; D010692:Phleomycins / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology; D014162:Transfection
D016054:DNA, Protozoan; D005819:Genetic Markers; D005839:Gentamicins; D010692:Phleomycins; D006921:Hygromycin B; D011691:Puromycin; C010680:antibiotic G 418