English
 
Help Privacy Policy Disclaimer
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT
  Transposon-mediated Chromsomal Integration of Transgenes in the Parasitic Nematode STrongyloids ratti and Establsihment of Stable Transgenic Lines

Shao, H., Li, X., Nolan, T. J., Massey, H. C. J., Pearce, E. J., & Lok, J. B. (2012). Transposon-mediated Chromsomal Integration of Transgenes in the Parasitic Nematode STrongyloids ratti and Establsihment of Stable Transgenic Lines. PLoS Pathogens, 8, e1002871. doi:10.1371/journal.ppat.1002871.

Item is

Basic

show hide
Genre: Journal Article

Files

show Files
hide Files
:
Shao et al..pdf (Publisher version), 2MB
Name:
Shao et al..pdf
Description:
-
Visibility:
Public
MIME-Type / Checksum:
application/pdf / [MD5]
Technical Metadata:
Copyright Date:
-
Copyright Info:
-
License:
-

Creators

show
hide
 Creators:
Shao, Hongguang1, Author
Li, Xinshe1, Author
Nolan, Thomas J.1, Author
Massey, Holman C. Jr.1, Author
Pearce, Edward J.2, Author              
Lok, James B.1, Author
Affiliations:
1External Organizations, ou_persistent22              
2Department Immunometabolism, Max Planck Institute of Immunobiology and Epigenetics, Max Planck Society, ou_2243648              

Content

show
hide
Free keywords: -
 Abstract: Genetic transformation is a potential tool for analyzing gene function and thereby identifying new drug and vaccine targets in parasitic nematodes, which adversely affect more than one billion people. We have previously developed a robust system for transgenesis in Strongyloides spp. using gonadal microinjection for gene transfer. In this system, transgenes are expressed in promoter-regulated fashion in the F1 but are silenced in subsequent generations, presumably because of their location in repetitive episomal arrays. To counteract this silencing, we explored transposon-mediated chromosomal integration of transgenes in S. ratti. To this end, we constructed a donor vector encoding green fluorescent protein (GFP) under the control of the Ss-act-2 promoter with flanking inverted tandem repeats specific for the piggyBac transposon. In three experiments, free-living Strongyloides ratti females were transformed with this donor vector and a helper plasmid encoding the piggyBac transposase. A mean of 7.9% of F1 larvae were GFP-positive. We inoculated rats with GFP-positive F1 infective larvae, and 0.5% of 6014 F2 individuals resulting from this host passage were GFP-positive. We cultured GFP-positive F2 individuals to produce GFP-positive F3 L3i for additional rounds of host and culture passage. Mean GFP expression frequencies in subsequent generations were 15.6% in the F3, 99.0% in the F4, 82.4% in the F5 and 98.7% in the F6. The resulting transgenic lines now have virtually uniform GFP expression among all progeny after at least 10 generations of passage. Chromosomal integration of the reporter transgenes was confirmed by Southern blotting and splinkerette PCR, which revealed the transgene flanked by S. ratti genomic sequences corresponding to five discrete integration sites. BLAST searches of flanking sequences against the S. ratti genome revealed integrations in five contigs. This result provides the basis for two powerful functional genomic tools in S. ratti: heritable transgenesis and insertional mutagenesis.

Details

show
hide
Language(s): eng - English
 Dates: 2012
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1371/journal.ppat.1002871
 Degree: -

Event

show

Legal Case

show

Project information

show

Source 1

show
hide
Title: PLoS Pathogens
  Other : PLoS Pathog.
Source Genre: Journal
 Creator(s):
Affiliations:
Publ. Info: San Francisco, CA : Public Library of Science
Pages: - Volume / Issue: 8 Sequence Number: - Start / End Page: e1002871 Identifier: ISSN: 1553-7366
CoNE: https://pure.mpg.de/cone/journals/resource/1000000000018830