Linked Life Data

9349274

Source:http://linkedlifedata.com/resource/pubmed/id/9349274

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
4
pubmed:dateCreated
1997-11-21
pubmed:databankReference
pubmed:abstractText
We report here on strategies aimed at improving the frequency of detectable recombination in plants by increasing the efficiency of selecting double-recombinants in transgenic calli. Gene targeting was approached on the Gln1 and the Pzfloci of Lotus japonicus, using Agrobacterium tumefaciens T-DNA replacement vectors. Large flanking regions, up to 22.9 kb, surrounding a positive selection marker were presented as substrates for homologous recombination. For easier detection of putative recombinants the negative selectable marker cytosine deaminase was inserted at the outside borders of the flanking regions offered for cross-over. A combination of positive and negative selection allowing double-recombinants to grow, while counter-selecting random insertions, was used to select putative targeting events. The more than 1000-fold enrichment observed with replacement vectors designed to minimize gene silencing demonstrated the efficiency of the negative selection. Using five different replacement vectors an estimated total of 18,974 transformation events were taken through the positive-negative selection procedure and 185 resistant calli obtained. Targeting events could not be verified in the survivors by PCR screening and Southern blot analysis. With this approach the frequency of detectable gene targeting in L. japonicus was below 5.3 x 10(-5), despite the large flanking sequences offered for recombination.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
0167-4412
pubmed:author
pubmed:issnType
Print
pubmed:volume
35
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
523-30
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1997
pubmed:articleTitle
Gene targeting approaches using positive-negative selection and large flanking regions.
pubmed:affiliation
Department of Molecular and Structural Biology, University of Aarhus, Denmark.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't