Linked Life Data

9572836

Source:http://linkedlifedata.com/resource/pubmed/id/9572836

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
18
pubmed:dateCreated
1998-6-8
pubmed:abstractText
The repeated modification of a genomic locus is a technically demanding but powerful strategy to analyze the function of a particular gene product or the role of cis-regulatory DNA elements in mammalian cells. The initial step is "tagging" a site with a selectable marker which is done by homologous recombination (HR) to modify a known locus or by random integration to study cis-regulatory elements at a reproducibly accessible genomic location. The tag is then used to target the construct of choice during an exchange step. Presented here is a novel technique in which the exchange is independent of HR and does not introduce vector sequences. It relies on our previous studies on the replacement of DNA cassettes by FLP-recombinase, whereby some common limitations can be overcome. To this end, the tag, a hygtk positive/negative selection marker, is integrated into the genome of embryonic stem (ES) cells. This marker is flanked by a wild-type Flp-recognition target (FRT) site on one end and by a modified heterospecific FRT site on the other. Successful Flp-mediated replacement of the hygtk cassette is enriched by ganciclovir (GANC) selection for cells that lack the encoded fusion protein. Thereby, the hygtk gene can be exchanged for virtually any sequence in a single efficient step without the need of introducing a positive selectable marker. The system can hence be used to analyze the function of either a gene product or regulatory sequences in ES cells or the transgenic mice derived thereof.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
5
pubmed:volume
37
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
6229-34
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1998
pubmed:articleTitle
DNA cassette exchange in ES cells mediated by Flp recombinase: an efficient strategy for repeated modification of tagged loci by marker-free constructs.
pubmed:affiliation
GBF, National Research Center for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't