Biolistic Transformation of Brugia Malayi

Abstract

Biolistics has become a versatile tool for direct gene transfer to various cell and tissue types. Following its successful use on the parasitic nematode Ascaris suum, we developed and evaluated biolistics in the transfection of the model filarial parasite Brugia malayi. Biolistics was proven to be an efficient strategy for transfection of all life stages of the parasite and paved the way for studies on elements essential for promoter function and gene regulation of filarial parasites. Here we present a biolistics protocol for the transfection of B. malayi based on the Biolistics PDS 1000/He system and gold microcarriers.

Transgenesis inStrongyloidesand related parasitic nematodes: historical perspectives, current functional genomic applications and progress towards gene disruption and editing

“…One such method with theoretical potential is biolistic transfer or particle bombardment. This method has been used widely for integrative transformation in C. elegans (Praitis, 2006; Schweinsberg and Grant, 2013), and for transient transformation of filariae (Higazi and Unnasch, 2013) and ascarid roundworms (Davis et al 1999). However, while this method has proven effective for transient expression of nucleic acid constructs, the level of mortality among bombarded parasitic nematodes has prevented its use thus far in derivation of viable, developing parasites that stably express transgenes (Xu et al 2011).…”

Functional analysis of microRNA activity in Brugia malayi

“…BmHSP70 (−394 to −1)/luc was included in the transfections as an internal standard to control for transfection efficiency. Transient transfections were performed as previously described (Higazi and Unnasch, 2013). In brief, embryos collected from five gravid adult female parasites were biolistically transfected with 1.35 mg of DNA-coated 0.6 ì m gold beads at a pressure of 1100 PSI using a BioRad PDS-1000/He™ System (BioRad Hercules, CA, USA).…”

Imaging trematode and nematode parasites

“…Transformation of parasitic nematodes and schistosomes with DNA or RNA constructs was first accomplished by particle bombardment (29)(30)(31)(32). Subsequently, methods for transfection of parasitic nematodes stressed microinjection of plasmid constructs into gonadal syncytia (20,33,34) or chemically mediated DNA transfer (35) in addition to particle bombardment (36). By contrast, electroporation (37)(38)(39) and transfection by pantrophic retroviruses (40) predominate along with particle bombardment as methods for gene transfer into schistosomes.…”

“…Heritable transformation and stained expression of both GFP and luciferase reporters have been achieved in S. mansoni (46,47). Likewise, Xu et al (35) achieved heritable transformation of B. malayi with a luciferase-based reporter, and experiments involving transient transfection by microinjection and particle bombardment indicate that GFP may also be expressed efficiently in this parasite (30,36). By contrast, only fluorescent reporters, GFP and mRFPmars, have been utilized to date in transgenic Strongyoides spp.…”