DNA CASSETTE EXCHANGE IN ES CELLS MEDIATED BY FLP RECOMBINASE - AN EFFICIENT STRATEGY FOR REPEATED MODIFICATION OF TAGGED LOCI BY MARKER-FREE CONSTRUCTS
J. Seibler et al., DNA CASSETTE EXCHANGE IN ES CELLS MEDIATED BY FLP RECOMBINASE - AN EFFICIENT STRATEGY FOR REPEATED MODIFICATION OF TAGGED LOCI BY MARKER-FREE CONSTRUCTS, Biochemistry, 37(18), 1998, pp. 6229-6234
The repeated modification of a genomic locus is a technically demandin
g but powerful strategy to analyze the function of a particular gene p
roduct 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 const
ruct of choice during an exchange step. Presented here is a novel tech
nique in which the exchange is independent of HR and does not introduc
e vector sequences. It relies on our previous studies on the replaceme
nt of DNA cassettes by FLP-recombinase, whereby some common limitation
s can be overcome. To this end, the tag, a hygtk positive/negative sel
ection marker, is integrated into the genome of embryonic stem (ES) ce
lls. 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 oth
er. Successful Flp-mediated replacement of the hygtk cassette is enric
hed by ganciclovir (GANC) selection for cells that lack the encoded fu
sion protein. Thereby, the hygtk gene can be exchanged for virtually a
ny 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 c
ells or the transgenic mice derived thereof.