Targeted integration into a rRNA locus results in uniform and high level expression of transgenes in Leishmania amastigotes

This report describes the construction of a DNA cassette for integration in to a genomic small sub-unit rRNA locus of Leishmania mexicana by homologous recombination. Reporter genes encoding beta-galactosidase or green fluores cent protein and the gene conferring hygromycin resistance were integrated downstream of a RNA polymerase I-driven rRNA promoter. To ensure high expre ssion of the marker proteins in the intracellular, amastigote stage, transg ene coding sequences were followed by the intergenic region of the L.. mexi cana cysteine proteinase B 2.8 gene which provides processing signals requi red for high level expression in this life-cycle stage. Integration of the DNA cassette was also efficiently obtained in L.. I,major. We show that eit her beta-galactosidase or the green fluorescent protein were abundantly, st ably and uniformly expressed in promastigotes and amastigotes of both Leish mania sp. The transgenic lines allow parasite detection at high sensitivity in the tissues of infected mice and will be useful to follow infections in macrophages in culture and in animal hosts. (C) 2000 Elsevier Science B.V. All rights reserved.