Linked Life Data

9566522

Source:http://linkedlifedata.com/resource/pubmed/id/9566522

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
1998-6-23
pubmed:abstractText
The hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) gene of the protozoan parasite Toxoplasma gondii encodes a safe, practical genetic marker suitable for both positive and negative selection. Taking advantage of the ability to control homologous versus nonhomologous recombination in haploid T. gondii tachyzoites by manipulating the length of homologous DNA sequence, we have explored the possibility of 'hit-and-run' mutagenesis to introduce gene knock-outs (or allelic replacements) at loci for which no known selection or screen is available. Using the uracil phosphoribosyl transferase (UPRT) locus as a target, a genomic clone containing approximately 8 kb encompassing the UPRT gene (but lacking essential coding sequence) was fused to a cDNA-derived HXGPRT 'minigene', which lacks sufficient contiguous genomic sequence for homologous recombination. After transfection of circular plasmid DNA, positive selection for HXGPRT activity identified stable transformants, > 30% of which were found to have integrated at the UPRT locus as 'pseudodiploids' (produced by single-site homologous recombination between the circular plasmid and genomic DNA). Upon removal of mycophenolic acid, resolution of pseudodiploids by spontaneous intrachromosomal homologous recombination was selected using 6-thioxanthine, yielding a 1:1 ratio of UPRT knock-out parasites to wild-type revertants, at frequencies of approximately 10(-6) per parasite doubling. Applications of 'hit-and-run' technology relative to other gene targeting strategies are discussed.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Mar
pubmed:issn
0166-6851
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
91
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
295-305
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1998
pubmed:articleTitle
Gene knock-outs and allelic replacements in Toxoplasma gondii: HXGPRT as a selectable marker for hit-and-run mutagenesis.
pubmed:affiliation
Department of Biology, University of Pennsylvania, Philadelphia 19104-6018, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.