Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression.

Abstract

To delineate segments of the genome of the human protozoan parasite Leishmania major necessary for replication and expression, we developed a vector (pR-NEO) which can be reproducibly introduced into L. major. This DNA was derived from a 30-kilobase extrachromosomal amplified DNA bearing the dihydrofolate reductase-thymidylate synthase gene, with the coding region for neomycin phosphotransferase substituted for that of dihydrofolate reductase-thymidylate synthase and a bacterial origin of replication and selectable marker added. G418-resistant lines were obtained at high efficiency by electroporation of pR-NEO (approaching 10-4 per cell), while constructs bearing an inverted neo gene or lacking Leishmania sequences did not confer resistance. pR-NEO replicated in L. major and gave rise to correctly processed transcripts bearing the trans-spliced miniexon. Molecular karyotype analysis showed that in some lines pR-NEO DNA exists exclusively as an extrachromosomal circle, a finding supported by the rescue of intact pR-NEO after transformation of Escherichia coli. These data genetically localize all elements required in cis for DNA replication, transcription, and trans splicing to the Leishmania DNA contained within pR-NEO DNA and signal the advent of stable transfection methodology for addressing molecular phenomena in trypanosomatid parasites.

An in vitro system for developmental and genetic studies of Leishmania donovani phosphoglycans

“…Parasite numbers were determined using a Coulter counter as previously described after an incubation of 2 days for the amastigotes and 3 days for the promastigotes. The effective concentration for 50% inhibition (EC 50 ) was defined as that drug concentration that resulted in a 50% decrease in cell number, measured at the time when control cultures lacking drug had reached 10 7 /ml [21].…”

“…Analysis of LPG expression showed that in general the primary transfectants did not make normal levels of LPG, for unknown reasons. Since episomal expression can be elevated by increasing the copy number [21], we used a combined FACS/drug selection protocol to recover parasites showing wild-type LPG expression. One clonal line (clone 2) of the primary lpg2 − /pSNBR-LmLPG2 transfectants was labeled with FITC-ricin, and the 5% of cells showing the most intense labeling were recovered by flow cytometry, and inoculated into 10-80 g/ml G418 in the presence of 2% galactose (first passage only).…”

“…Cosmid vectors are used commonly in functional genetic rescue studies in Leishmania. We tested two electroporation conditions, a 'low voltage' protocol used commonly for Leishmania [21] and a recent modification termed the 'high voltage' protocol [26]. Parasites were electroporated as either promastigotes or amastigotes in the presence of cosmid B1660, and plated on homologous growth medium containing 100 or 300 g hygromycin B/ml (promastigotes or amastigotes, respectively).…”

Functional genetic identification of PRP1, an ABC transporter superfamily member conferring pentamidine resistance in Leishmania major

“…Promastigotes were grown in M199 medium supplemented as described [11,22]. Parasites from late log phase cultures were transfected by electroporation (500 F, 2.25 kV/cm) using 20-40 g of cosmid DNA.…”

Successful transient introduction of leishmania RNA virus into a virally infected and an uninfected strain of Leishmania

“…Transfection of Leishmania with foreign genes has been reported both transiently (11) and stably (12,13 (16) supplemented with 5% fetal bovine serum and 1% fresh, filter-sterilized human urine (17).…”