Figure 1.
Schematic diagram of the principle of the iKO system.
(A) Two components of the iKO system, a KO line and a TIGRE line. (B) Generation of iKO mouse by crossing the KO and TIGRE lines. The iKO is a composite mouse in which: 1) both copies of the endogenous Gene X are disrupted by the insertion; 2) rtTA is driven by the promoter of Gene X; and 3) the cDNA of Gene X is in the TIGRE locus under the control of TRE. Functional Gene X is only expressed from the TIGRE locus when rtTA is bound to Dox, and therefore is inducibly and reversibly regulated by Dox.
Figure 2.
Retroviral vector used to search for tightly regulated loci and strategy to introduce a new gene into these predetermined loci.
(A) Structure of the pRTonZ retroviral vector. To prevent effects of viral enhancer on the TRE promoter, the enhancer sequence was deleted in the 3′ long terminal repeat (LTR). Subsequent transduction into target cells is expected to lead to enhancer deletion in both LTRs. The insert was cloned in the opposite orientation of the LTRs, so that the polyA addition signals would not decrease the viral titer. (B) Excision of the lacZ reporter gene from TIGRE loci. LoxP-flanked sequences within the retroviral vector are removed by transient expression of Cre recombinase in ES cells, leaving a single copy of loxP site in the genome. ES cells become G418-sensitive since the loxneo gene loses its promoter and initiating AUG. (C) Introduction of a new gene into the TIGRE locus. The G418-sensitive ES cells selected in (B) are cotransfected with the TIGRE-targeting vector carrying a new gene (gene X) and the Cre expression vector. Proper Cre-mediated recombination between the TIGRE-targeting vector, containing the new gene, and the TIGRE site introduces the new gene into the TIGRE locus and converts the G418 sensitive ES cells into G418 resistant (the expected recombinant leads to neo expression by placing the PGK promoter and initiating AUG upstream of the loxneo gene). Symbols are: pAgl, rabbit β-globin gene polyA addition signal; P, mouse phosphoglycerate kinase-1 gene promoter; AUG, initiating AUG of neomycin phosphotransferase gene; loxneo, neomycin phosphotransferase gene having loxP sequence in-frame to initiating AUG; pABGH, bovine growth hormone gene polyA addition signal; G418r, G418 resistant; G418s, G418 sensitive.
Figure 3.
Characterization of gene regulation at TIGRE loci.
(A) Screening for optimal integration sites in ES cells and classification of ES clones by X-gal staining. A representative ES clone is shown for each class with and without phase contrast for better imaging of ES cell morphology and X-gal staining, respectively. (B) Screening of class I clones for high inducibility. Forty three class I ES clones were transfected with a tTA expression vector and β-gal activity was quantified 48 hours post-transfection. Bars: open, without tTA; filled, with tTA. A luciferase expression vector was cotransfected to normalize β-gal activities. Three ES clones were designated as T1, T2 and T3 as shown in the figure, and were used to generate mice. (C) Gene regulation in mice generated from tightly regulated ES clones. Three mouse strains were established from ES clones T1, T2 and T3, and crossed to MMTV-tTA mouse. Top three panels: β-gal activity was measured in the following three genotypes: nontransgenic mice (lacZ(−)tTA(−), open bars); mice with lacZ gene but without tTA (lacZ(+)tTA(−), lightly shaded bars); mice with both lacZ gene and tTA gene (lacZ(+)tTA(+), filled bars). Values are shown as means with error bars of standard deviations from five male and five female animals. Values of nontransgenic mice (open bars) are common in all three panels. Bottom panel: tTA mRNA expression level quantified by real-time PCR from one male and one female mouse. Mean values are presented by filled bars. Note that values are shown in logarithmic scale.
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. Clone numbers correspond to those in Figure S1. 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. Clone numbers correspond to those in Figure S2. (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 αCaMKII-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 αCaMKII-tTA, β-gal staining is seen in the same regions αCaMKII-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).
Figure 5.
Chromosomal location of the T1 TIGRE locus.
(A) Genomic sequences surrounding the T1 TIGRE locus. The underlined AAAG sequence was duplicated upon viral integration and the viral TIGRE vector was inserted exactly in between the duplication. (B) BLAT search of the UCSC Mouse Genome Browser (http://genome.ucsc.edu/cgi-bin/hgBlat) with genomic sequences (as in (A)) surrounding T1 revealed the localization of T1 locus to chr9 qA3. This panel is a screen shot of the BLAT search result. The location of the sequences used for the search is indicated by a vertical bar next to “YourSeq”. The insertion site is in between two genes: AB124611 and Carm1 (alternative name Prmt4), and does not seem to disrupt either gene.
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.
Figure 7.
Plasma cholesterol levels and atherosclerotic lesion progression/regression regulated by Dox in ApoE iKO mice.
(A) Plasma cholesterol levels in the ApoE iKO, KO and WT group mice in the absence and presence of Dox. (Littermate mice of various genotypes other than homozygous KO or iKO, i.e. ApoE+/+, ApoE+/−, ApoE+/+;TRE-ApoE and ApoE+/−;TRE-ApoE, all displayed normal and indistinguishable blood cholesterol levels, and never developed lesions under any treatment regime used in our study. Consequently they were lumped together as the “WT group”.) Plasma cholesterol levels were first measured in mice fed with normal food without Dox (−Dox) and both ApoE iKO and KO mice showed significantly higher cholesterol levels compared to WT group mice (p = 0.35 between iKO and KO, p<0.0001 between iKO and WT or between KO and WT, Student's t-test). The mice were then switched to Dox-containing food, and plasma cholesterol levels were measured again 4 days (+Dox 4d) and 7 days (+Dox 7d) later. Cholesterol level of ApoE iKO mice dropped to WT levels in less than 4 days while that of ApoE KO remained high (p<0.0001 between iKO and KO or between KO and WT, p = 0.86 between iKO and WT). Sometime later, some Dox-treated mice were switched back to normal food, and plasma cholesterol levels were measured again 4 days (−Dox 4d) and 7 days (−Dox 7d) after Dox withdrawal. Cholesterol level of ApoE iKO mice significantly elevated by day 4 (p<0.001 between iKO and WT, p<0.01 between iKO and KO) and approached pre-Dox treatment level by day 7 (p<0.0001 between iKO and WT, p = 0.13 between iKO and KO). ***P<0.001. (B) Atherosclerotic lesion progression. Aortas were stained with Sudan IV to visualize the lesions in red. The arch region of the aorta contains the most extensive areas of lesions and is shown here. A group of iKO, KO and WT mice were treated with Dox-containing food starting before the onset of lesions, and were compared with mice fed with normal food. At 7 months of age, aortas were dissected from these mice and lesions were examined. ApoE iKO mice showed extensive aortic lesions as the KO mice in the absence of Dox, and yet no lesions at all as the WT mice in the presence of Dox. (C) Atherosclerotic lesion regression. ApoE iKO and KO mice of 5 months of age were switched from normal food to Dox-containing food. Aortic lesions were examined before (at 5 months) and after (at 9 months) the Dox treatment. After 4 months of Dox treatment, the lesions in KO mice continued to grow, whereas in the iKO mice the lesions had regressed. (D) Quantification of the aortic atherosclerotic lesion areas in the arch region above the first intercostal artery, as expressed by the percentage of lesion areas versus the whole aortic area in this segment. All genotypes are matched with ages for different Dox treatment. Dox-treated groups are: iKO+Dox: Dox food started at 2–4 months of age (before the onset of atherosclerosis); iKO+Dox regression: Dox food started at 5–6 months of age (after the onset of atherosclerosis); KO+Dox: Dox food started at 2–4 months of age (before the onset of atherosclerosis). The results are compared using one-way ANOVA followed by Neuman-Keul's post hoc test. Both iKO+Dox and iKO+Dox regression groups had significantly reduced atherosclerotic areas compared to the remaining groups. *P<0.05, **P<0.01. The iKO mice without Dox, as well as KO mice either with or without Dox, all developed comparable areas of lesions (p>0.05 in all pair-wise comparisons). The iKO mice treated with Dox before the onset of atherosclerosis had nearly no lesions and were significantly different from the above groups (p<0.01 iKO+Dox versus iKO−Dox; p<0.01 iKO+Dox versus KO−Dox; p<0.05 iKO+Dox versus KO+Dox). The iKO mice treated with Dox after the onset of atherosclerosis had significantly reduced atherosclerotic areas compared to iKO mice without Dox or KO mice either with or without Dox (p<0.05 in all comparisons between iKO+Dox regression versus iKO−Dox, KO−Dox or KO+Dox).