An Inducible and Reversible Mouse Genetic Rescue System - Genetic Engineering: Recent Developments in Applications - page 254

Figure 6. Characterization of gene regulation in iKO mice. (A) Construction and genomic structure of the
ApoE iKO mice. Endogenous ApoE gene comprises of 4 exons. In the ApoE KO line, retroviral vector is
inserted into the third intron of the ApoE gene, 205 bp upstream of the fourth exon (the largest coding exon).
The retroviral vector contains the virus backbone (including 5 ′ LTR and 3 ′ LTR), a splice acceptor (SA) - stop
codon (stop) - IRES cassette immediately followed by rtTA, a PGK promoter (P) driven neo selection marker
flanked by two loxP sites (L), and a transcriptional terminator sequence (t). pA, polyadenylation sequence. From
this locus, transcription initiated from the endogenous ApoE gene continues through rtTA to form an ApoE-
SA-Stops-IRES-rtTA-polyA hybrid transcript in place of the full-length endogenous ApoE transcript. The rtTA
protein is produced from this hybrid transcript through IRES-mediated translation, and in turn turns on the
expression of the TRE-ApoE from the TIGRE locus only when Dox is present. The ApoE TIGRE locus contains
an exogenous copy of ApoE cDNA driven by TRE and flanked by four copies of chicken β -globin insulator
(ins) sequences, two on each side, and a PGK promoter (P) driven loxneo selection marker. (B) Side-by-side
comparison of the expression of rtTA and each individual endogenous gene in various tissues by semi-quantitative
RT-PCR. Three GPCR genes are shown here: P2Y6, RE2 and LGR6. Heterozygous mice are used so that the
endogenous transcripts from the WT allele and the rtTA-containing hybrid transcripts from the KO allele can
be amplified from the same RNA preps. Each RT-PCR reaction (RT+) has an RT- control run simultaneously
to exclude any possibility of genomic DNA contamination. (C) Comparison of three transcripts by RT-PCR
from the same RNA prep of the liver, the major site of normal ApoE production, of the ApoE+/−;TRE-ApoE
mice - endogenous ApoE transcript (endoApoE), TRE-ApoE transcript from the TIGRE locus (TRE-ApoE)
and ApoE-rtTA hybrid transcript (endoApoE-rtTA). Mice were fed either with or without Dox. As expected,
expression of endoApoE and rtTA were independent of Dox. However, expression of TRE-ApoE was strictly
dependent on Dox - it was undetectable in its absence and significantly expressed in its presence, indicating high
degree of ApoE regulation achieved in the mice.