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. 2008 May 9;4(5):e1000069. doi: 10.1371/journal.pgen.1000069

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This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.

This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.

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(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.