Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
. 2017 Mar;144(3):327-342.
doi: 10.1017/S0031182016000391. Epub 2016 Mar 22.

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

J B Lok  1 , H Shao  1 , H C Massey  1 , X Li  1
Affiliations
Review

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

J B Lok et al. Parasitology. 2017 Mar.

Abstract

Transgenesis for Strongyloides and Parastrongyloides was accomplished in 2006 and is based on techniques derived for Caenorhabditis elegans over two decades earlier. Adaptation of these techniques has been possible because Strongyloides and related parasite genera carry out at least one generation of free-living development, with adult males and females residing in soil contaminated by feces from an infected host. Transgenesis in this group of parasites is accomplished by microinjecting DNA constructs into the syncytia of the distal gonads of free-living females. In Strongyloides stercoralis, plasmid-encoded transgenes are expressed in promoter-regulated fashion in the F1 generation following gene transfer but are silenced subsequently. Stable inheritance and expression of transgenes in S. stercoralis requires their integration into the genome, and stable lines have been derived from integrants created using the piggyBac transposon system. More direct investigations of gene function involving expression of mutant transgene constructs designed to alter intracellular trafficking and developmental regulation have shed light on the function of the insulin-regulated transcription factor Ss-DAF-16. Transgenesis in Strongyloides and Parastrongyloides opens the possibility of powerful new methods for genome editing and transcriptional manipulation in this group of parasites. Proof of principle for one of these, CRISPR/Cas9, is presented in this review.

Keywords: Caenorhabditis; Strongyloides; CRISPR/Cas9; chromosomal integration; microinjection; nematode; transgenesis; transposon.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
_target specific insertional mutagenesis in S. stercoralis via CRISPR. (A) Basic scheme involving transformation of P0 free-living female worms with plasmids pPV549 encoding Cas9, codon optimized for the extremely AT-rich genome of S. stercoralis (Massey et al. ; Hunt et al. 2016), pPV567 encoding sgRNA specific for exon 5 of Ss-daf-16, pPV576 encoding the 24 bp insert sequence, containing stop codons in all reading frames, flanked by 50 bp homology arms complementary to homology regions HR1 and HR2, respectively, in the _target and copies of _target Sequence 2 for excision by CRISPR and pPV570 encoding _target 2-specific sgRNA. (B) Detail of _target for double stranded break (DSB) in Ss-daf-16 followed by incorporation of insert via homology directed repair (HDR). Insert contains a novel forward priming site F1. Priming sites R0-R2 in flanking genomic region for nested PCR assay are indicated, with expected sizes of products. (C) PCR products (arrows at right) from nested PCR reactions involving primer pairs F1-R1 and F1-R2. Data are from two biological replicates (EXP-1, EXP-2) with control reactions using wild type gDNA (Ss Wt) and no-template controls (NTC). Note concordance with expected product sizes. The primary reaction with F1-R0 yielded no detectable products, but did so when concentrated and used in subsequent nested reactions. (D) Sequences of Ss-daf-16 _target region (blue characters) and insert (green highlight) with homology arms (yellow highlight) and of cloned products of F1–R1 and F1–R2 reactions showing insert and with homology arm and 5′ flanking genomic sequence (unhighlighted black characters). GenBank accession numbers for constructs: pPV549 – KU665998, pPV567 – KU665999, pPV570 – KU666000, pPV576 – KU666001.
Fig. 2.
Fig. 2.
Partial regulation of gfp expression in S. stercoralis by fusion to the trimethoprim (TMP)-stabilized DHFR degradation domain. (A) Bicistronic vector pPV505 encoding gfp and mRFPmars with intervening self-cleaving p2a domain, all under the body wall-specific Ss-act-2 promoter. (B–D) DIC, red and green channel images, respectively, of a post free-living first-stage larva expressing pPV505. Note uniform green and red fluorescence. (E) Bicistronic vector pPV512 identical to pPV505 except encoding gfp/DHFR-DD fusion. (F–H) Images of a post free-living first-stage larva expressing pPV512 and cultured without TMP. Note diminished green fluorescence. (I–K) images of an early post free-living third-stage larva expressing pPV512 and cultured with 50 µm TMP. Note partial restoration of green fluorescence (K). (H) Green fluorescence quantitation in larvae expressing pPV512 and cultured with or without TMP. Means and SEM for 7 worms per sample; *Significant (P = 0·046) difference from ‘No TMP’ control. GenBank accession numbers for constructs: pPV505 – KU665996, pPV512 – KU665997.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ashton F. T., Bhopale V. M., Holt D., Smith G. and Schad G. A. (1998). Developmental switching in the parasitic nematode Strongyloides stercoralis is controlled by the ASF and ASI amphidial neurons. Journal of Parasitology 84, 691–695. - PubMed
    1. Balu B., Shoue D. A., Fraser M. J. Jr. and Adams J. H. (2005). High-efficiency transformation of Plasmodium falciparum by the lepidopteran transposable element piggyBac. Proceedings of the National Academy of Sciences of the United States of America 102, 16391–16396. - PMC - PubMed
    1. Beckmann S. and Grevelding C. G. (2012). Paving the way for transgenic schistosomes. Parasitology 139, 651–668. - PubMed
    1. Britton C., Samarasinghe B. and Knox D. P. (2012). Ups and downs of RNA interference in parasitic nematodes. Experimental Parasitology 132, 56–61. - PubMed
    1. Cahill C. M., Tzivion G., Nasrin N., Ogg S., Dore J., Ruvkun G. and Alexander-Bridges M. (2001). Phosphatidylinositol 3-kinase signaling inhibits DAF-16 DNA binding and function via 14-3-3-dependent and 14-3-3-independent pathways. Journal of Biological Chemistry 276, 13402–13410. - PubMed

Publication types

LinkOut - more resources

  NODES
Note 4
twitter 2