An Inducible and Reversible Mouse Genetic Rescue System - Genetic Engineering: Recent Developments in Applications - page 249

Those loci were further examined in mice. Three independent TRE-lacZ
mouse lines were generated from class I ES clones T1, T2 and T3 (Figure 3C).
Heterozygous TRE-lacZ mice were crossed to MMTV-tTA mice, which has been
reported to be transcriptionally active in a wide variety of cell types [11], allowing
for the examination of lacZ induction in various tissues. Three genotypes of mice
(lacZ(−)tTA(−), lacZ(+)tTA(−), and lacZ(+)tTA(+)) were analyzed for β -gal activ-
ity (Figure 3C). Activity of lacZ(−)tTA(−) represents endogenous eukaryotic β -gal
activity. Difference between lacZ(−)tTA(−) and lacZ(+)tTA(−) indicates basal ac-
tivity of lacZ gene in the absence of tTA, and comparison of lacZ(+)tTA(−) and
lacZ(+)tTA(+) reveals induction levels in the presence of tTA. Overall, the three
mouse lines showed similar pattern of β -gal activity, although some differences
were also seen. Basal activities were low but detectable in many tissues, and they
were comparable to the values measured in the parental ES clones. β -gal activity
was inducible in almost every tissue, and overall induction levels correlated with
the expression levels of tTA (Figure 3C, bottom panel).
improvement of Gene regulation control at the tiGre Loci
Although the three loci T1, T2 and T3 showed tight regulation of gene expression,
basal activity was still detectable in many tissues. This could result from enhancers
in the vicinity of the integration sites. To solve this problem, we flanked the TRE-
lacZ reporter by the insulator sequence derived from the chicken β -globin locus
[12],[13]. The insulator sequences were introduced into all three loci (T1, T2, T3)
by Cre-mediated recombination according to the scheme shown in Figures 2B and
2C and regulation of the lacZ gene was examined by transient expression of tTA
in ES cells (Figure 4A). The insulators reduced basal β -gal activity to levels indis-
tinguishable from wild type ES cells. In contrast, inducibility was not impaired by
the insulator sequence, indicating its effectiveness for increasing stringency of gene
regulation. The insulators were also introduced into class II, III and IV ES clones,
which showed high basal activity (Figure 3A). Although the insulator sequences
were effective, basal activity was still clearly detectable in every clone (Figure 4B).
To test whether tight regulation is achieved by using other genes, we replaced the
lacZ gene with a luciferase gene. With this reporter, insulators reduced basal ac-
tivity by 14, 20 and 11 fold in T1, T2 and T3 loci, respectively, bringing it close
to the instrument detection limit (Figure 4C). Calculation of the number of lu-
ciferase molecule per ES cell (Supporting Methods) revealed that on average only
1, 0.7 and 0.3 luciferase molecule were expressed per cell in T1, T2 and T3 loci,
respectively (Figure 4D). Importantly, induced levels were not impaired by the
insulator (Figure 4C), leading to high induction ratio of luciferase activity. Similar
basal activity levels could also be achieved by expressing transrepressor. However,
our system is simpler because no additional protein expression is required.