An Inducible and Reversible Mouse Genetic Rescue System - Genetic Engineering: Recent Developments in Applications - page 255

tightly regulated Apoe expression and blood cholesterol
Levels by dox in the Apoe iKo Mice
To prove the concept of iKO system, we generated ApoE iKO mice by creating
ApoE KO line and ApoE TIGRE line separately and breeding them together (Fig-
ure 6A), and attempted to model human conditions such as hypercholesterolemia
and atherosclerosis. ApoE KO line was created by screening the mutant ES cell
library as mentioned above. ApoE TIGRE line was created by inserting a TRE-
ApoE transgene, flanked with 4 copies of chicken β -globin insulators, into the
T1 TIGRE locus. RT-PCR analysis of heterozygous mice (ApoE+/−; TRE-ApoE)
showed that expression of TRE-ApoE was strictly dependent on Dox (Figure 6C).
Real-time qPCR using primers specific for the endogenous ApoE or TRE-ApoE
(2 sets of primers for each gene) showed that endogenous ApoE mRNA level is
very high (compared to 18s rRNA), and the TRE-ApoE mRNA level in the pres-
ence of Dox is ~6.7 fold lower than the endogenous ApoE ( ∆ Ct = 2.7 between
TRE-ApoE and endogenous ApoE), while in the absence of Dox TRE-ApoE is
>55,000 fold lower than the endogenous ApoE ( ∆ Ct>15.8). This shows the TRE-
ApoE induction by Dox is >8,000 fold.
Next, we carried out phenotypic analysis of homozygous ApoE iKO mice
(ApoE−/−; TRE-ApoE), i.e. mice having both endogenous ApoE alleles inactivat-
ed and carrying TRE-ApoE in the TIGRE locus. We analyzed blood cholesterol
levels in these mice in the absence and presence of Dox. Constitutive KO (i.e.
ApoE−/− without TRE-ApoE in the TIGRE locus) and WT group mice that were
littermates of iKO were used as controls. As shown in Figure 7A, in the absence
of Dox the iKO mice had high cholesterol levels similar to that of the KO mice;
in the presence of Dox, the iKO showed normal cholesterol levels, demonstrating
that inducible expression of ApoE can lead to the reversion of the KO phenotype
of hypercholesterolemia. When Dox was withdrawn, the cholesterol levels in the
iKO mice rose again. These on/off switches occurred rapidly, within a few days
after Dox administration or withdrawal.
dox-regulated Atherosclerosis Progression and regression in
the Apoe iKo Mice
We examined the atherosclerotic lesion formation in the aortas of the ApoE iKO
mice. As controls we used KO and WT group mice that were littermates of iKO.
In the absence of ApoE protein, aortic atherosclerotic lesions start to form around
3-4 months of age and progress continually with time. One set of iKO, KO and
WT group mice were treated with Dox-containing food throughout their life, and
were compared with mice fed with normal food. Figure 7B shows the atherosclerotic