Improvements in transfection efficiency and tests of RNA interference (RNAi) approaches in the protozoan parasite Leishmania

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

“…The efficiencies obtained were comparable to those obtained previously with promastigotes of an avirulent derivative of L. donovani 1S [28], and with constructs smaller than the cosmid tested here the efficiency can be as high as 3%/electroporated cell [26]. In previous studies we showed that the transient transfection efficiency of LdB promastigotes was also high (∼20%; [26]). These high efficiencies are of great value in a variety of genetic approaches, including functional genetic rescue with genomic DNA libraries in cosmids or other vectors [43].…”

“…2). The high-voltage electroporation conditions were superior for amastigotes as well as for promastigotes, giving on average 5-8-fold more colonies under equivalent conditions, as seen previously with smaller reporter constructs [26]. Increasing amounts Fig.…”

“…2). This represented an efficiency of nearly 0.1% per cell electroporated, about 10-fold less than obtained by transfection of promastigotes with smaller constructs [26].…”

“…For efficiency tests, two different electroporation conditions were used: "low voltage" (500 F, 450 V, 2 mm cuvette; 2.25 kV/cm) or "high voltage" (25 F, 1500 V, 4 mm cuvette; 3.75 kV/cm); [26]. The test DNA was cosmid B1660, which contains the L. major H region inserted into the shuttle vector cLHYG [27,28].…”

“…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).…”