Biomedical Engineering Reference
In-Depth Information
reciprocal DNA recombination between two loxP sites of the same directionality. Thus,
a sequence with flanking loxP sites (floxed) will be excised by site-specific recombi-
nation in the presence of Cre, leaving a single loxP site marking the point of excision
(Figure 10.1c). By crossing a line of mice with a floxed allele generated by gene target-
ing and a line of transgenic mice expressing Cre under the control of a tissue-specific
promoter, the allele will be deleted specifically in that tissue. In an alternative approach,
conditional knockin of point mutations has also been reported [36]. The repertoire of
mice expressing Cre recombinase under different endogenous, tissue and developmen-
tal stage-specific promoters is continually growing. Viral delivery of Cre to specific
tissues may be useful if a suitable Cre line of mice is not available. It is important
that the endogenous gene functions normally until recombined by Cre; therefore, target-
ing modifications must be made without interfering with coding, splicing or regulatory
regions. Similar to the generation of null mutants, the loxP sites must be inserted in
such a way that recombination will render the gene inactive, avoiding compensatory
alternative splicing.
In order to approach as close to normal expression as possible, it is advisable to remove
the drug selection marker by designing FRT sites flanking the selection cassette. Trans-
fection of ES cells with an FLP-expressing vector or crossing chimeras with transgenic
mice expressing FLP in the germline effects excision of the cassette.
In some cases, deletion of the entire gene is desired, especially in cases of complicated
alternative splicing, which may be the case for some large genes. Cre recombinase can
also mediate large genomic deletions between two loxP sites located at a great distance.
Two separate targeting vectors with different positive selection markers should be used
for insertion of two loxP sites by homologous recombination at the 5' and 3' proximal
regions of a target gene. At least one TK gene should also be introduced in one of the
targeting constructs. Correctly targeted clones are then transfected with a Cre-expressing
plasmid, and clones with deletion, and thus TK deletion, are selected for by resistance to
FIAU. Alternatively, targeting vectors can be designed that carry two complementary but
non-functional fragments of a positive selection marker. Upon Cre-mediated excision,
the two fragments are brought together and drug resistance is restored. Using these
methods, deletions of several hundred kilobase pairs can be achieved. Further discussion
of conditional and inducible techniques is found elsewhere [37, 38].
4
Inducible deletion
: Inducible mutation strategies using tetracycline or steroid recep-
tor antagonists have been successful in achieving genetic manipulations at a desired
stage of development, and can also provide tissue specificity [38]. Tet on/off systems
allow switching on and off of gene expression through activation or inhibition of the
tetracycline-dependent transactivating (tTA) factor with tetracycline analogs such as
doxycycline. tTA binds to the tetO operator sequence placed upstream of a minimal
promoter and the gene of interest through gene targeting. This is an appropriate strategy
for reversible gene expression changes, but can often have 'leaky' gene expression. The
Cre-ER
T2
-tamoxifen system is a recommended approach for inducible and irreversible
gene deletion [39, 40]. When fused to a mutant estrogen receptor ligand binding domain
that is activated by tamoxifen but not estrogen, Cre remains in the cytoplasm and cannot
catalyze recombination events. On addition of tamoxifen, the specific ligand for ER
T2
,
the Cre-ER
T2
fusion protein translocates to the nucleus, where it now mediates deletion
of floxed alleles. Thus, deletion is controlled temporally by tamoxifen administration and
spatially by tissue-specific Cre-ER
T2
expression. Tamoxifen availability can be limiting,
