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to WT levels in less than 4 days while that of ApoE KO remained high (p<0.0001 between iKO and KO or
between KO and WT, p = 0.86 between iKO and WT). Sometime later, some Dox-treated mice were switched
back to normal food, and plasma cholesterol levels were measured again 4 days (−Dox 4d) and 7 days (−Dox 7d)
after Dox withdrawal. Cholesterol level of ApoE iKO mice significantly elevated by day 4 (p<0.001 between iKO
and WT, p<0.01 between iKO and KO) and approached pre-Dox treatment level by day 7 (p<0.0001 between
iKO and WT, p = 0.13 between iKO and KO). ***P<0.001. (B) Atherosclerotic lesion progression. Aortas were
stained with Sudan IV to visualize the lesions in red. The arch region of the aorta contains the most extensive
areas of lesions and is shown here. A group of iKO, KO and WT mice were treated with Dox-containing food
starting before the onset of lesions, and were compared with mice fed with normal food. At 7 months of age,
aortas were dissected from these mice and lesions were examined. ApoE iKO mice showed extensive aortic
lesions as the KO mice in the absence of Dox, and yet no lesions at all as the WT mice in the presence of Dox.
(C) Atherosclerotic lesion regression. ApoE iKO and KO mice of 5 months of age were switched from normal
food to Dox-containing food. Aortic lesions were examined before (at 5 months) and after (at 9 months) the
Dox treatment. After 4 months of Dox treatment, the lesions in KO mice continued to grow, whereas in the iKO
mice the lesions had regressed. (D) Quantification of the aortic atherosclerotic lesion areas in the arch region
above the first intercostal artery, as expressed by the percentage of lesion areas versus the whole aortic area in this
segment. All genotypes are matched with ages for different Dox treatment. Dox-treated groups are: iKO+Dox:
Dox food started at 2-4 months of age (before the onset of atherosclerosis); iKO+Dox regression: Dox food
started at 5-6 months of age (after the onset of atherosclerosis); KO+Dox: Dox food started at 2-4 months of
age (before the onset of atherosclerosis). The results are compared using one-way ANOVA followed by Neuman-
Keul's post hoc test. Both iKO+Dox and iKO+Dox regression groups had significantly reduced atherosclerotic
areas compared to the remaining groups. *P<0.05, **P<0.01. The iKO mice without Dox, as well as KO mice
either with or without Dox, all developed comparable areas of lesions (p>0.05 in all pair-wise comparisons).
The iKO mice treated with Dox before the onset of atherosclerosis had nearly no lesions and were significantly
different from the above groups (p<0.01 iKO+Dox versus iKO−Dox; p<0.01 iKO+Dox versus KO−Dox; p<0.05
iKO+Dox versus KO+Dox). The iKO mice treated with Dox after the onset of atherosclerosis had significantly
reduced atherosclerotic areas compared to iKO mice without Dox or KO mice either with or without Dox
(p<0.05 in all comparisons between iKO+Dox regression versus iKO−Dox, KO−Dox or KO+Dox).
We further investigated what happens if ApoE protein expression is turned on
after the atherosclerotic lesions have already formed. ApoE iKO and KO mice of
5 months of age were switched from normal food to Dox-containing food for the
next 4 months. Aortic atherosclerotic lesions were examined before (at 5 months)
and after (at 9 months) the Dox treatment. As shown in Figure 7C, at 5 months
of age, both iKO and KO had developed similar levels of lesions. After 4 months
of Dox treatment, the lesions in KO mice continued to grow, whereas in the
iKO mice, the lesions had regressed nearly completely with only scar-like tissues
remaining, suggesting that the lipid-containing foam cells have disappeared from
the lesions. The results were verified by quantification and statistical analysis of
the lesions by one-way ANOVA followed by Neuman-Keul's post hoc test (Figure
7D).
discussion
We have developed a reversible and inducible rescue system for gene KO in mice
and have applied this method into the ApoE gene. Our system complements ex-
isting inducible gene expression approaches and provides certain advantages. The
tamoxifen-dependent Cre-ERT2 [19],[20] recombination can drive inducible
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