title: Fluorescent Leishmania species: development of stable GFP expression and its application for in vitro and in vivo studies.
abstract: Reporter genes have proved to be an excellent tool for studying disease progression. Recently, the green fluorescent protein (GFP) ability to quantitatively monitor gene expression has been demonstrated in different organisms. This report describes the use of Leishmania tarentolae (L. tarentolae) expression system (LEXSY) for high and stable levels of GFP production in different Leishmania species including L. tarentolae, L. major and L. infantum. The DNA expression cassette (pLEXSY-EGFP) was integrated into the chromosomal ssu locus of Leishmania strains through homologous recombination. Fluorescent microscopic image showed that GFP transgenes can be abundantly and stably expressed in promastigote and amastigote stages of parasites. Furthermore, flow cytometry analysis indicated a clear quantitative distinction between wild type and transgenic Leishmania strains at both promastigote and amastigote forms. Our data showed that the footpad lesions with GFP-transfected L. major are progressive over time by using fluorescence small-animal imaging system. Consequently, the utilization of stable GFP-transfected Leishmania species will be appropriate for in vitro and in vivo screening of anti-leishmanial drugs and vaccine development as well as understanding the biology of the host-parasite interactions at the cellular level.
authors.affiliation: Molecular Immunology and Vaccine Research Laboratory, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Azam
authors.initials: A
authors.lastname: Bolhassani
authors.forename: Tahereh
authors.initials: T
authors.lastname: Taheri
authors.forename: Yasaman
authors.initials: Y
authors.lastname: Taslimi
authors.forename: Soheila
authors.initials: S
authors.lastname: Zamanilui
authors.forename: Farnaz
authors.initials: F
authors.lastname: Zahedifard
authors.forename: Negar
authors.initials: N
authors.lastname: Seyed
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Torkashvand
authors.forename: Behrouz
authors.initials: B
authors.lastname: Vaziri
authors.forename: Sima
authors.initials: S
authors.lastname: Rafati
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002469:Cell Separation; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004195:Disease Models, Animal; D005260:Female; D005434:Flow Cytometry; D015870:Gene Expression; D017125:Genes, Protozoan; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D007091:Image Processing, Computer-Assisted; D007891:Leishmania / Q000235:genetics / Q000378:metabolism*; D018314:Leishmania infantum / Q000235:genetics / Q000378:metabolism; D018320:Leishmania major / Q000235:genetics / Q000378:metabolism; D007896:Leishmaniasis / Q000469:parasitology*; D008264:Macrophages / Q000469:parasitology*; D051379:Mice; D008807:Mice, Inbred BALB C; D030781:Organisms, Genetically Modified; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019076:Transgenes
chemical_list: D049452:Green Fluorescent Proteins