Strongyloides stercoralis: cell- and tissue-specific transgene expression and co-transformation with vector constructs incorporating a common multifunctional 3' UTR

Abstract

Transgenesis is a valuable methodology for studying gene expression patterns and gene function. It has recently become available for research on some parasitic nematodes, including Strongyloides stercoralis. Previously, we described a vector construct, comprising the promoter and 3' UTR of the S. stercoralis gene Ss era-1 that gives expression of GFP in intestinal cells of developing F1 progeny. In the present study, we identified three new S. stercoralis promoters, which, in combination with the Ss era-1 3' UTR, can drive expression of GFP or the red fluorescent protein, mRFPmars, in tissue-specific fashion. These include Ss act-2, which drives expression in body wall muscle cells, Ss gpa-3, which drives expression in amphidial and phasmidial neurons and Ss rps-21, which drives ubiquitous expression in F1 transformants and in the gonads of microinjected P0 female worms. Concomitant microinjection of vectors containing GFP and mRFPmars gave dually transformed F1 progeny, suggesting that these constructs could be used as co-injection markers for other transgenes of interest. We have developed a vector "toolkit" for S. stercoralis including constructs with the Ss era-1 3' UTR and each of the promoters described above.

Fig. 1

Neuron-specific expression of gfp under the Ss gpa-3 promoter in S. stercoralis transformed with construct pAJ09 (Table 1). A, B. DIC and fluorescence images, respectively, of a transformant L1 showing GFP localization in amphidial cell bodies (Amph), in their dendritic processes leading into the amphidial channel and in phasmidial neurons (Ph), C, D. high-magnification DIC and fluorescence images, respectively, of specimen in panels A–B showing GFP localization in nerve ring (NR) and 8 amphidial cell bodies (CB). Scale bars: A = 50 μM; C = 20 μM.

Fig. 2

Body wall-specific expression of gfp and mRFPmars under the Ss act-2 promoter in S. stercoralis transformed with construct pAJ08 and pAJ50, respectively (Table 1). A, B. DIC and fluorescence images, respectively, showing GFP localization in F1 transformant first-stage larvae (L1) transformed with the reporter construct pAJ08. C, D. DIC and fluorescence images, respectively, showing mRFPmars localization in F1 transgenic L1 transformed with the reporter construct pAJ50. All scale bars = 50 μM.

Fig. 3

Ubiquitous expression of gfp under the Ss rps-21 promoter in S. stercoralis transformed with construct pAJ20 (Table 1). A, B. DIC and fluorescence images, respectively showing ubiquitous distribution of GFP in a vermiform embryo transformed with pAJ20. C, D. a pAJ20 transformant first-stage larva showing gfp expression in virtually all body tissues with the highest level of expression occurring in the genital primordium (GP). E, F. Composite DIC and fluorescence images, respectively, of a P0 free-living female microinjected with pAJ20. Note the high level of gfp-expression in the gonad and eggs. Scale bars in A and C = 20 μM; E = 100 μM.

Fig. 4

Expression of S. stercoralis-based reporter constructs in C. elegans. A–D. Variable expression patterns of gfp expression under the Ss act-2 promoter in C. elegans transiently transformed with construct pAJ08 (Table 1). D. DIC and fluorescence overlay image showing GFP localization in the sixth pharyngeal muscle ring (pm6). Scale bar for A–D = 50 μM. E–H. Images of gfp expression under the Ss rps-21 promoter in C. elegans transformed with construct pAJ20 (Table 1). Images show GFP localization in glial cells in the amphidial (E, F) and phasmidial (G, H) complexes. Scale bars in E and G = 20μM.

Fig 5

Co-transformation of S. stercoralis with dual reporter constructs, pAJ09 and pAJ50 (Table 1). A, B. DIC and merged red and green fluorescence images, respectively, of an F1 transgenic S. stercoralis L1 expressing gfp under the Ss gpa-3 promoter (pAJ09) in amphidial neurons and nerve ring and mRFPmars under the Ss act-2 promoter (pAJ50) in body wall muscles. Scale bar = 30 μM.

Fig. 6

Photomontage depicting expression of gfp under the Ss act-2 promoter in an F1 parasitic female transformed with construct pAJ08 (Table 1) and recovered at necropsy from an experimentally infected gerbil. A, B. Composite DIC and fluorescence images, respectively. Scale bar = 100 μM.