Transgenesis in the parasitic nematode Strongyloides ratti

Abstract

Strongyloides and related genera are advantageous subjects for transgenesis in parasitic nematodes, primarily by gonadal microinjection as has been used with Caenorhabditis elegans. Transgenesis has been achieved in S. stercoralis and in Parastrongyloides trichosuri, but both of these lack well-adapted, conventional laboratory hosts in which to derive transgenic lines. By contrast, Strongyloides ratti develops in laboratory rats with high efficiency and offers the added advantages of robust genomic and transcriptomic databases and substantial volumes of genetic, developmental and immunological data. Therefore, we evaluated methodology for transgenesis in S. stercoralis as a means of transforming S. ratti. S. stercoralis-based GFP reporter constructs were expressed in a proportion of F1 transgenic S. ratti following gonadal microinjection into parental free-living females. Frequencies of transgene expression in S. ratti, ranged from 3.7% for pAJ09 to 6.8% for pAJ20; respective frequencies for these constructs in S. stercoralis were 5.6% and 33.5%. Anatomical patterns of transgene expression were virtually identical in S. ratti and S. stercoralis. This is the first report of transgenesis in S. ratti, an important model organism for biological investigations of parasitic nematodes. Availability of the rat as a well-adapted laboratory host will facilitate derivation of transgenic lines of this parasite.

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

“…In an initial study (Li et al 2011), two transcriptional reporter constructs were tested in S. ratti . These fused promoters for either the cellular actin-encoding gene Ss-act-2 or the ribosomal small subunit-encoding gene Ss-rps-21 to the gfp coding sequence with both constructs incorporating the Ss-era-1 3′UTR.…”

“…To date, reporter constructs used in all but one study of anatomical gene expression patterns in Strongyloides and Parastrongyloides have constituted simple fusions of coding sequences of LacZ (Grant et al 2006 a ), gfp (Grant et al 2006 a ; Li et al 2006, 2011; Junio et al 2008; Massey et al 2013; Yuan et al 2014 a , b ) or egfp (Stoltzfus et al 2012 a , 2014) to putative promoters for the genes of interest. In contrast to these, a study employing transgenesis to investigate the function of Ss-daf-16 (previously named fktf-1 ) employed a construct designed to express an N-terminal fusion of GFP with wild-type Ss -DAF-16b (Castelletto et al 2009).…”

Chemosensory behaviors of parasites

“…The molecular basis of chemoreception in parasitic nematodes has not yet been investigated. However, recent breakthroughs in the development of techniques for stable genetic transformation of S. stercoralis and S. ratti [46, 47] should enable new avenues of research into the molecular mechanisms that underlie chemosensation in these parasites.…”

Heritable genetic transformation of Strongyloides stercoralis by microinjection of plasmid DNA constructs into the male germline

“…The body plans of free-living strongyloidoid females are strikingly similar to those of C. elegans hermaphrodites (Lints and Hall, 2009) with amphidelphic ovaries that have syncytial zones in their distal ends (Schad, 1989; Kulkarni et al, 2016), and this has permitted adaptation of microinjection techniques for gene transfer in C. elegans to strongyloidoid parasites with few modifications (Lok and Massey, 2002; Grant et al, 2006; Lok, 2006, 2012; Lok et al, 2016). The result has been a reliable system for introducing transgenes via the female germlines of S. stercoralis (Li et al, 2006; Junio et al, 2008) and Strongyloides ratti (Li et al, 2011) that are expressed in a promoter-regulated fashion in F1 progeny, and ultimately may be integrated into the chromosomes of S. ratti and inherited and expressed indefinitely through host and culture passage (Shao et al, 2012). …”

“…1A), were designed to express the GFP and a red fluorescent protein (mRFPmars), respectively, under the promoter for Sr-eft-3 in S. ratti . Just as regulatory sequences from S. stercoralis give active transgene expression in S. ratti (Li et al, 2011), the Sr-eft-3 promoter drives a high level of reporter expression in S. stercoralis from transgenes delivered via the female germline (Fig. 1E).…”