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 into a genomic small sub-unit rRNA locus of Leishmania mexicana by homologous recombination. Reporter genes encoding beta-galactosidase or green fluorescent protein and the gene conferring hygromycin resistance were integrated downstream of a RNA polymerase I-driven rRNA promoter. To ensure high expression of the marker proteins in the intracellular, amastigote stage, transgene coding sequences were followed by the intergenic region of the L. mexicana cysteine proteinase B 2.8 gene which provides processing signals required for high level expression in this life-cycle stage. Integration of the DNA cassette was also efficiently obtained in L. major. We show that either beta-galactosidase or the green fluorescent protein were abundantly, stably and uniformly expressed in promastigotes and amastigotes of both Leishmania 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.
Misslitz
A
A
Max-Planck Institut für Biologie, Abteilung Membranbiochemie, Tübingen, Germany.
Mottram
J C
JC
Overath
P
P
Aebischer
T
T
D000818:Animals; D005260:Female; D005434:Flow Cytometry; D015870:Gene Expression; D020459:Genes, rRNA; D049452:Green Fluorescent Proteins; D018320:Leishmania major / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D007894:Leishmania mexicana / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D016773:Leishmaniasis, Cutaneous / Q000469:parasitology; D008164:Luminescent Proteins / Q000378:metabolism; D008264:Macrophages; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D016053:RNA, Protozoan / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics; D011995:Recombination, Genetic; D019076:Transgenes; D001616:beta-Galactosidase / Q000378:metabolism
D008164:Luminescent Proteins; D016053:RNA, Protozoan; D012335:RNA, Ribosomal; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase