In vivo transfection of developmentally competent Brugia malayi infective larvae

Abstract

Transient transfection of isolated Brugia malayi embryos by biolistics has proven to be useful in defining promoter structure and function in this parasite. However, isolated transfected embryos are developmentally incompetent. A method of producing developmentally competent transfected parasites is therefore needed. We report that L3 parasites can be chemically transfected in situ in the peritoneal cavity of a gerbil with a construct consisting of a secreted luciferase reporter gene containing a promoter, the 3' untranslated region and first intron derived from the B. malayi 70kDa heat shock protein gene. The in situ chemically transfected parasites are developmentally competent, producing adult parasites with an efficiency similar to that obtained from implanted untreated L3s. Cultured adult parasites and progeny microfilariae (mf) derived from L3s transfected with this construct secreted luciferase into the culture medium. When the transfected mf were fed to mosquitoes and the resulting L3s collected, the L3s also secreted luciferase into the culture medium. Progeny mf from transgenic adult parasites contained transgenic DNA, and the transgenic mRNA produced in these parasites was found to be correctly cis-and trans-spliced. In situ chemical transformation thus results in developmentally competent transfected B. malayi in which the transgenic sequences remain transcriptionally active in all life cycle stages and are present in the subsequent generation.

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

“…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). Also, the low frequency of transgene integration into the chromosomes of C. elegans following biolistic transfer can be offset by a selection process in which worms with a mutation in unc-119 that impairs motility and dauer formation are rescued by a plasmid encoding wild-type unc-119 co-transferred with the construct of interest (Schweinsberg and Grant, 2013).…”

The development of RNA interference (RNAi) in gastrointestinal nematodes

“…(Junio et al 2008) may allow knock-out or knock-in of specific pathway genes to identify the limiting steps. Recently, Xu et al (2011) reported chemical transfection of infective L3 larvae of B. malayi in situ, with subsequent stable expression of a luciferase reporter gene. Generation of transgenic parasites therefore has the potential to enhance RNAi silencing mechanisms and also provide a means of expressing dsRNA from hairpin constructs which may overcome the limitations of environmental RNAi.…”

Isotype analysis of gerbil–mouse heterohybridomas by RT-PCR

“…Mongolian gerbils (Meriones unguiculatus) are used in biomedical research, such as in studies of infections with Helicobacter pylori (Wu et al, 2008) and Brugia malayi (Xu et al, 2011). Because of the absence of mAbs specific to gerbil Igs, antibodies in gerbil sera have been measured by agglutinin assay (Kai et al, 2002) and polyclonal antibodies (pAbs) to mouse immunoglobulin (Ig) isotypes showing cross-reactivity to their gerbil counterparts (Amorim et al, 2010).…”