Successful transgenesis of the parasitic nematode Strongyloides stercoralis requires endogenous non-coding control elements.
Critical investigations into the cellular and molecular biology of parasitic nematodes have been hindered by a lack of modern molecular genetic techniques for these organisms. One such technique is transgenesis. To our knowledge, the findings reported here demonstrate the first heritable DNA transformation and transgene expression in the intestinal parasite Strongyloides stercoralis. When microinjected into the syncitial gonads of free-living S. stercoralis females, a construct fusing the S. stercoralis era-1 promoter, the coding region for green fluorescent protein (gfp) and the S. stercoralis era-1 3' untranslated region was expressed in intestinal cells of normally developing F1 transgenic larvae. The frequency of transformation and GFP expression among F1 larvae was 5.3%. By contrast, expression of several promoter::gfp fusions incorporating only Caenorhabditis elegans regulatory elements was restricted to abortively developing F1 embryos of S. stercoralis. Despite its lack of regulated expression, PCR revealed that one of these C. elegans-based vector constructs, the sur-5::gfp fusion, is incorporated into F1 larval progeny of microinjected female worms and then transmitted to the F2 through F5 generations during two host passages conducted without selection and punctuated by free-living generations reared in culture. Heritable DNA transformation and regulated transgene expression, as demonstrated here for S. stercoralis, constitute the essential components of a practical system for transgenesis in this parasite. This system has the potential to significantly advance the molecular and cellular biological study of S. stercoralis and of parasitic nematodes generally.
Li
Xinshe
X
Department of Pathobiology, School of Veterinary Medicine and University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104-6050, USA.
Massey
Holman C
HC
Nolan
Thomas J
TJ
Schad
Gerhard A
GA
Kraus
Kelly
K
Sundaram
Meera
M
Lok
James B
JB
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics; D018509:DNA, Helminth / Q000235:genetics; D005260:Female; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D017238:Genes, Helminth; D005849:Gerbillinae; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008845:Microinjections / Q000379:methods; D008969:Molecular Sequence Data; D018510:RNA, Helminth / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D017171:Strongyloides stercoralis / Q000235:genetics* / Q000378:metabolism
D018509:DNA, Helminth; D018510:RNA, Helminth; D049452:Green Fluorescent Proteins