Culture for genetic manipulation of developmental stages of Schistosoma mansoni

Abstract

SUMMARY Genomes of the major human helminth parasites, and indeed many others of agricultural significance, are now the research focus of intensive genome sequencing and annotation. A draft genome sequence of the filarial parasite Brugia malayi was reported in 2007 and draft genomes of two of the human schistosomes, Schistosoma japonicum and S. mansoni reported in 2009. These genome data provide the basis for a comprehensive understanding of the molecular mechanisms involved in schistosome nutrition and metabolism, host-dependent development and maturation, immune evasion and invertebrate evolution. In addition, new potential vaccine candidates and drug targets will likely be predicted. However, testing these predictions is often not straightforward with schistosomes because of the difficulty and expense in maintenance of the developmental cycle. To facilitate this goal, several developmental stages can be maintained in vitro for shorter or longer intervals of time, and these are amenable to manipulation. Our research interests focus on experimental studies of schistosome gene functions, and more recently have focused on development of transgenesis and RNA interference with the longer term aim of heritable gene manipulation. Here we review methods to isolate and culture developmental stages of Schistosoma mansoni, including eggs, sporocysts, schistosomules and adults, in particular as these procedures relate to approaches for gene manipulation. We also discuss recent advances in genetic manipulation of schistosomes including the deployment of square wave electroporation to introduce reporter genes into cultured schistosomes.

Reversible paralysis of Schistosoma mansoni by forchlorfenuron, a phenylurea cytokinin that affects septins

“…Cercariae were obtained by shedding infected snails under bright light for 2 h at 23 °C. Adult worms were cultured under 5% CO 2 at 37 °C in DMEM, supplemented with 10% FBS and 1×penicillin/streptomycin as described (Dalton et al, 1997; Mann et al, 2009; Zeraik et al, 2013). …”

Vaccinomics for the Major Blood Feeding Helminths of Humans

“…Parasitic helminths, by virtue of their often complex life cycles and large genomes, have rendered themselves refractory to many genetic manipulation tools that allow the exploration of gene function (Mann et al, , 2010. RNAi is now widely used to assess gene function in schistosomes, and appears to be particularly effective for genes expressed in tissues readily accessible to dsRNA, such as the tegument and gastrodermis (Krautz-Peterson et al, 2010;Stefanic et al, 2010).…”

Whole-genome sequence of Schistosoma haematobium

“…Given the challenges in propagating these parasites, particularly S. haematobium, in the laboratory 29 , the ability to sequence the genome from a single pair of worms represents a important step in characterizing the genomes of a diverse range of other schistosomes and neglected tropical disease pathogens, including food-borne flukes, and toward addressing fundamental and controversial questions regarding their genetics, evolution, ecology, epidemiology, pathogenesis and host-parasite relationships. The genome provides a solid foundation for future large-scale and integrated studies of gene function and essentiality, using tools such as RNA interference and transgenesis 28 , and will also facilitate urgently needed proteomic explorations.…”