Generating Transgenics and Knockouts in Strongyloides Species by Microinjection.

Abstract

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and Strongyloides ratti. S.stercoralis is a human-parasitic, skin-penetrating nematode that infects approximately 610 million people, while the rat parasite S.ratti is closely related to S.stercoralis and is often used as a laboratory model for S.stercoralis. Both S.stercoralis and S.ratti are easily amenable to the generation of transgenics and knockouts through the exogenous nucleic acid delivery technique of intragonadal microinjection, and as such, have emerged as model systems for other parasitic helminths that are not yet amenable to this technique. Parasitic Strongyloides adults inhabit the small intestine of their host and release progeny into the environment via the feces. Once in the environment, the larvae develop into free-living adults, which live in feces and produce progeny that must find and invade a new host. This environmental generation is unique to the Strongyloides species and similar enough in morphology to the model free-living nematode Caenorhabditis elegans that techniques developed for C. elegans can be adapted for use with these parasitic nematodes, including intragonadal microinjection. Using intragonadal microinjection, a wide variety of transgenes can be introduced into Strongyloides. CRISPR/Cas9 components can also be microinjected to create mutant Strongyloides larvae. Here, the technique of intragonadal microinjection into Strongyloides, including the preparation of free-living adults, the injection procedure, and the selection of transgenic progeny, is described. Images of transgenic Strongyloides larvae created using CRISPR/Cas9 mutagenesis are included. The aim of this paper is to enable other researchers to use microinjection to create transgenic and mutant Strongyloides.