Linked Life Data

9657336

Source:http://linkedlifedata.com/resource/pubmed/id/9657336

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
1998-10-22
pubmed:abstractText
We have engineered a mutant version of the green fluorescent protein GFP (Cormack et al. Selected for bright fluorescence in E. coli. Gene 1996;173:33-38) for expression in the protozoan parasite Toxoplasma gondii. Although intact GFP was not expressed at any detectable level, GFP fusion proteins could be detected by fluorescence microscopy, flow cytometry (FACS), and immunoblotting. Both extracellular tachyzoites and T. gondii-infected host cells could readily be sorted by FACS, which should facilitate a variety of selection strategies. Several selectable markers were tested for their ability to produce stable green transgenic parasites. Fluorescence intensity was directly correlated with gene copy number and protein expression level. Weak selectable markers such as chloramphenicol acetyl transferase (CAT) driven by the SAG1 promoter, which yield multicopy insertions, are therefore most effective for selecting green fluorescent parasites-particularly when coupled to constructs which employ a strong promoter to drive GFP expression. Transformation vectors developed in the course of this work should be of general utility for the overexpression of heterologous transgenes in Toxoplasma. CAT-GFP fusion proteins were expressed in the parasite cytoplasm. GFP fusions to the P30 major surface antigen (linked on the same plasmid to a CAT selectable marker under control of various promoters) could be detected in dense granules within living cells, and were efficiently secreted into the parasitophorous vacuole. GFP fusions to the rhoptry protein ROP1 were targeted to rhoptries (specialized secretory organelles at the apical end of the parasite).
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
0166-6851
pubmed:author
pubmed:issnType
Print
pubmed:day
1
pubmed:volume
92
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
325-38
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1998
pubmed:articleTitle
Expression, selection, and organellar targeting of the green fluorescent protein in Toxoplasma gondii.
pubmed:affiliation
Department of Biology, University of Pennsylvania, Philadelphia 19104-6018, USA. striepen@sas .upenn.edu
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't