An Inducible and Reversible Mouse Genetic Rescue System - Genetic Engineering: Recent Developments in Applications - page 251

To evaluate the insulator efect in vivo, we also generated mice containing
TRE-lacZ gene and insulators, or TRE-luciferase (Luc) gene and insulators at the
TIGRE locus (T1), and bred them with mice containing tTA under the control
of various promoters. Basal expression was examined in multiple tissues of TRE-
Luc mice by RT-PCR (Figure 4E). Luc mRNA was undetectable in all tissues
examined except testis, indicating very low basal levels of expression throughout
the body. To examine if the Luc transcript detected in testis can produce func-
tional Luc proteins, we conducted luciferase activity assays from protein extracts
of all these tissues. he luciferase activity in testis was at the similar low basal level
as all other tissues examined (data not shown) as well as the original TIGRE ES
clone containing TRE-Luc with insulators that had been shown to express ap-
proximately one Luc molecule per cell (Figure 4D), suggesting that the RT-PCR
band detected in testis resulted from aberrant transcription that did not gener-
ate functional protein. To examine induced expression, we used lines of mice
with tTA under the control of two brain-speciic promoters: α -CaMKII (cal-
cium/calmodulin-dependent protein kinase II) [14] or NSE (neuron-speciic eno-
lase) [15]. Sagital sections of 50 µm thickness across the entire brain from TRE-
lacZ, PCAMKII-tTA/TRE-lacZ or PNSE-tTA/TRE-lacZ mice were stained for
β -galactosidase (gal) activity and representative sections are shown in Figure 4F. In
TRE-lacZ mice, no β -gal staining was seen in any parts of the brain. In PCAM-
KII-tTA/TRE-lacZ or PNSE-tTA/TRE-lacZ mice, intense β -gal staining was ob-
served in speciic regions of the brain deined by the two promoters respectively.
hese results demonstrate tight control of the TIGRE locus in animals.
characterization of the tiGre Locus
Using the splinkerette PCR method [16], we obtained genomic fragments cover-
ing either 5 ′ or 3 ′ junctions of the TIGRE vector insertion site in the T1 ES cell
line. After sequencing the fragments, we determined the precise integration site of
the viral TIGRE vector in the T1 TIGRE locus, which is located on chromosome
9. Genomic sequences surrounding the T1 TIGRE locus are shown in Figure 5A.
Characteristic of retrovirus insertions, four nucleotides immediately adjacent to
the insertion were duplicated and the viral TIGRE vector was inserted exactly in
between the duplication. BLAT search of the UCSC Mouse Genome Browser
with genomic sequences surrounding T1 revealed the localization of T1 locus to
chr9 qA3 (Figure 5B). The insertion site is flanked by two genes: AB124611 and
Carm1. The insertion site is located 3 ′ to the hypothetical gene AB124611 with
unknown function and undetermined polyA site. The insertion site is also ~1.5 kb
upstream of the transcriptional start of Carm1. Carm1 is ubiquitously expressed
and Carm1−/− mice are embryonic lethal [17]. However, we have not observed
overt developmental or other abnormalities in heterozygous or homozygous T1