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Figure 4. Tightening of gene regulation at TIGRE loci by insulators. (A) Insulator effect in class I clones.
Parental ES clones without the insulator (T1, T2, T3) and clones with the insulator (T1inZ, T2inZ, T3inZ)
were transfected by tTA expression vector, and β -gal activity was measured 48 hours post-transfection. Three
independent integrant clones with insulator were analyzed in each integration site. A luciferase expression vector
was cotransfected to normalize the β -gal activities. WT, wild type ES cells. Bars: open, without tTA; filled, with
tTA. The same symbol was used in (B) and C). (B) Insulator effect on basal β -gal activity in class II, III and IV
clones. Insulator sequence was introduced into three ES clones categorized in class II, III and IV of Figure 3A.
Note that values are shown in logarithmic scale. (C) Insulator effect on the regulation of luciferase gene. The
lacZ gene of the three class I clones (T1, T2, T3) were replaced by luciferase gene without (T1L, T2L, T3L) or
with (T1inL, T2inL, T3inL) the insulator. Two independent integrants were established in each case. A lacZ
expression vector was cotransfected to normalize the luciferase activities. (D) Number of luciferase molecule
per cell in class I clones with the insulator sequence. (E) RT-PCR of the luciferase (Luc, upper panel) and
positive control GAPDH (lower panel) transcripts in a TIGRE line (T1) with TRE-Luc and insulators. Each
tissue has an RT-PCR reaction (RT+, left lane) and an RT- control (right lane) run simultaneously to exclude
any possibility of genomic DNA contamination. The last lane is a positive control of genomic DNA PCR just
for Luc. (F) β -gal staining of brain sagital sections (50 µm) of mice carrying a TRE-LacZ (+insulators) TIGRE
(T1) line alone (top panel) or combined with either α CaMKII-tTA (middle panel) or NSE-tTA (bottom panel)
transgene. There is no detectable β -gal staining in the absence of inducer (top panel). When TRE-LacZ TIGRE
line is combined with α CaMKII-tTA, β -gal staining is seen in the same regions α CaMKII-tTA is expressed
- mainly cortex, hippocampus and striatum (middle panel). When TRE-LacZ TIGRE line is combined with
NSE-tTA, β -gal staining is seen in the same regions NSE-tTA is expressed - mainly striatum, dentate gyrus and
cerebellum (bottom panel).
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