Biology Reference
In-Depth Information
genomic locus. It has the advantage of, 1) ability to turn genes on or of at will by
adding or removing doxycycline (Dox) at any time during the animal's life, thus
minimizing embryonic lethality, developmental effects, and compensatory effects;
2) high degree of regulation to any gene inserted at the TIGRE locus, which has
been selected to confer minimal basal expression and high inducibility, and to in-
sert any gene of interest in a single step by Cre/loxP recombination; 3) efficiency;
the design allowing streamlined production of both KO and TIGRE mice makes
it possible to generate iKOs for a large number of genes in a cost-effective manner;
4) flexibility; KO and TIGRE lines can be engineered independently and com-
bined in numerous ways, making a wide range of applications possible.
As a proof of concept, we report the characterization of an iKO of the apo-
lipoprotein E gene (ApoE iKO). ApoE plays a key role in regulating cholesterol
metabolism and atherosclerosis progression. ApoE KO mice develop hypercho-
lesterolemia and atherosclerosis that closely resemble the human conditions and
are rapidly reversed when APOE protein is supplied [3],[4],[5],[6],[7],[8]. Thus,
inducible and reversible regulation of ApoE expression could result in rapid physi-
ological changes, which in turn can help assess the iKO technology. Furthermore,
the phenotype of ApoE deficiency is quantifiable and very sensitive to leaky ex-
pression, allowing for the evaluation of the stringency of gene regulation by iKO
technology [9]. Here we demonstrate that in the ApoE iKO mice, ApoE gene
expression, as well as blood cholesterol levels, is tightly controlled by Dox. In
the presence of Dox, ApoE is expressed and the cholesterol levels are low; in its
absence, the reverse is observed. Furthermore, on examination of aortic athero-
sclerosis in the ApoE iKO mice we found that Dox treatment before the onset
of atherosclerotic lesions completely prevented lesion formation and Dox treat-
ment after extensive lesions had already formed resulted in regression of the le-
sions. These results demonstrate the reversibility of the iKO, leading to phenotype
switching within the same animal. ApoE iKO is also useful in its own right as a
novel model system for the study of molecular mechanisms underlying atheroscle-
rosis progression and regression.
results
Principle of the iKo system
As illustrated in Figure 1A, two genetically modified mouse strains are created.
The first is a KO line in which a Tet-dependent transactivator (rtTA in this ex-
ample) is inserted into the target gene (Gene X). The insertion inactivates Gene
X, and places rtTA under the control of the endogenous promoter of Gene X.
The KO line can be generated via either homologous recombination or insertional
Search WWH ::
Custom Search