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i.e. basal expression level was reduced to less than one luciferase molecule per
cell without impairing inducibility (Figure 4C, D). Insulators were also shown
previously to improve inducibility of randomly integrated TRE-reporters [33]. It
should be noted that in our study the insulator effect was limited in many other
loci (Figure 4A versus 4B), demonstrating the uniqueness of the TIGRE loci. The
genomic location of the T1 TIGRE locus, which was most extensively used in our
study including the ApoE iKO mice, was determined (Figure 5). Further manipu-
lation of this locus would be possible to expand its application.
The stringent gene regulation is demonstrated in the ApoE iKO mice. It is
known that regulation of blood cholesterol levels is very sensitive to the plasma
ApoE protein levels. Even with the production of 3% of wild-type level of ApoE
protein, the hypercholesterolemia and atherosclerosis phenotypes of the ApoE
KO mice can be reversed [9]. Given this high sensitivity, the fact that our ApoE
iKO mice in the uninduced state (i.e. in the absence of Dox) exhibit similarly high
levels of cholesterol compared to ApoE KO mice indicates that there is hardly any
expression of functional APOE. Reversing the KO at will is a particularly power-
ful approach in atherosclerotic regression studies. Dox-treatment of ApoE iKO
mice results in expression of ApoE, marked reduction of plasma cholesterol levels,
and regression of aortic atherosclerotic lesions. These findings are consistent with
previous studies showing that aggressive lipid lowering or expression of ApoE
can induce regression of pre-existing atherosclerotic lesion [7],[8],[34],[35]. It
is becoming increasingly clear that lesion regression is regulated by a complex
interplay between lipids, inflammation and the immune system [35]. The ApoE
iKO mice will allow detailed studies on the roles of specific genes in these complex
interactions.
The binary nature of the iKO system is inherently simple, with the KO line
serving dual roles: it could be used as a constitutive KO or combined with the
TIGRE line to produce an inducible and reversible iKO. The 10-million clone ES
cell library we utilized [18] has been estimated to contain insertional mutations
for >90% of genes, and individual ES clones with retroviral insertions in a specific
target gene can be rapidly identified through a PCR pooling strategy and subse-
quently isolated in a streamlined process. The identification and modification of
the TIGRE locus allows rapid insertion of any gene of interest via co-transfection
with Cre. Therefore, each component of the binary system, the KO or the TIGRE
line, is amenable for high-throughput production to generate inducible and re-
versible KOs for a large number of genes.
It should be noted that the iKO system may not be applicable in certain situa-
tions where highly stringent gene regulation is required. For example, even though
the system had enough stringency in low basal activity and high induction, the
induced gene expression level is usually not exactly the same as the endogenous
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