Very recently, standard chemical transformation methods were applied to the transfection of moulting L3 B. malayi parasites (92). Previously, biolistic and microinjection techniques have been attempted in these parasites (93,94); however, both techniques are invasive and, in particular, biolistics adversely affected either the viability of the transgenic parasites or the ability of isolated embryos to undergo further development. In contrast, chemical transformation resulted in developmentally competent transfected parasites, and Selleck Erastin the reporter gene used was transcriptionally active throughout all stages of the parasites’ life cycle. However, transgene expression
was significantly reduced compared, for example, to transgenes introduced by biolistics, and hence, alternative chemical or biological methods of delivery into B. malayi are being investigated. It is clear that techniques for the delivery of exogenous genes into parasitic helminths are now considered well established. However, in all examples described thus far, enforced transgene expression has been largely restricted to reporter genes such as GFP and luciferase, unlike with RNAi approaches where many genes involved in diverse cellular pathways have been targeted. Nevertheless, the full potential of transgenesis in parasitic helminths is starting to be realized for the study of parasite protein function. For example,
the role of the protein forkhead transcription factor-1 isoform b (FKTF-1b) in S. stercoralis’
infective larval development was recently Panobinostat molecular weight investigated using GFP-linked proteins, including dominant negative mutants, introduced BCKDHA into adult female parasites via intra-gonadal microinjection (95). Here, the authors showed that recombinant FKTF-1b tagged with GFP localizes to specific tissues remodelled in infective larvae. Furthermore, mutant forms of FKTF-1b designed to interfere with endogenous FKTF-1b function resulted in incomplete development of the infective larval structures and prevented some transgenic larvae from arresting in the infective stage, indicating that FKTF-1b is required for the proper development of S. stercoralis’ infective larvae. Some of the first clear insights into the possibility of achieving heritable transgenesis were made in S. stercoralis and Parastrongyloides trichosuri. Li et al. (96) described the transgenesis of reporter constructs into the syncitial gonads of free-living females by microinjection. The plasmid constructs were found to be transmitted for as many as five generations, but were eventually lost without selection. However, none of the constructs were expressed beyond F2; hence, a stable transgene-expressing line was not generated. Greater success was achieved with P. trichosuri where transgene-expressing worms were established and maintained as transgenic worm lines (97).