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. 2018 Jul 6;8(3):78-86.
doi: 10.1556/1886.2018.00008. eCollection 2018 Sep 28.

Humanization of the Blood-Brain Barrier Transporter ABCB1 in Mice Disrupts Genomic Locus - Lessons From Three Unsuccessful Approaches

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Free PMC article

Humanization of the Blood-Brain Barrier Transporter ABCB1 in Mice Disrupts Genomic Locus - Lessons From Three Unsuccessful Approaches

Markus Krohn et al. Eur J Microbiol Immunol (Bp). .
Free PMC article

Abstract

ATP-binding cassette (ABC) transporters are of major importance for the restricted access of toxins and drugs to the human body. At the body's barrier tissues like the blood-brain barrier, these transporters are highly represented. Especially, ABCB1 (P-glycoprotein) has been a priority target of pharmaceutical research, for instance, to aid chemotherapy of cancers, therapy resistant epilepsy, and lately even neurodegenerative diseases. To improve translational research, the humanization of mouse genes has become a popular tool although, like recently seen for Abcb1, not all approaches were successful. Here, we report the characterization of another unsuccessful commercially available ABCB1 humanized mouse strain. In vivo assessment of transporter activity using positron emission tomography imaging revealed a severe reduction of ABCB1 function in the brain of these mice. Analyses of brain mRNA and protein expression showed that the murine Abcb1a gene is still expressed in homozygous humanized animals while expression of the human gene is minimal. Promoter region analyses underpinned that the introduced human gene might dysregulate normal expression and provided insights into the regulation of both transcription and translation of Abcb1a. We conclude that insertion of the human coding DNA sequence (CDS) into exon 3 instead of exon 2 most probably represents a more promising strategy for Abcb1a humanization.

Keywords: ABC transporter; ABCB1; P-gp; PET imaging; humanization; mouse models.

Conflict of interest statement

Conflict of Interest The authors have no conflict of interest to report.

Figures

Figure 1.
Figure 1.
ABCB1 transport activity at the BBB measured with (R)-[C]verapamil PET imaging. Brain uptake of (R)-[C]verapamil expressed as brain-to-blood radioactivity concentration ratio (Kb,brain) at 60 min after radiotracer injection in female C57BL/6 mice (veh: n = 6, tariquidar: n = 5), hABCB1 mice (genOway; veh: n = 3, tariquidar: n = 3), and hABCB1 mice (Taconic; veh: n = 4, tariquidar: n = 3) treated with vehicle solution (2.5% [w/v] aq. dextrose solution) or tariquidar (15 mg/kg body weight) at 2 h before radiotracer injection. For comparison, Kb,brain in vehicle-treated Abcb1a/b(–/–) mice (n = 4) is also shown [36]. Data are mean ± standard deviation. Statistical significance was determined by 2-way ANOVA with Bonferroni post-hoc test. **p < 0.01; ***p < 0.001
Figure 2.
Figure 2.
Relative mRNA expression of Abcb1a and ABCB1. Human ABCB1 mRNA is expressed at very low levels in hABCB1 mice only. No decrease of Abcb1a mRNA expression was detected in hABCB1 mice compared to wild-type animals. Shown are the relative expression of mouse Abcb1a (green) and human ABCB1 (blue) mRNA in male wild-type (n = 7) and hABCB1 (n = 10) mice based on the comparison of ct values. Ct values were normalized to mouse Actb mRNA expression using 2(–Δct) calculation. Data are mean ± standard deviation. Statistical significance was determined by 1-way ANOVA with Tukey's honest significant difference post-hoc test. ****p < 0.0001
Figure 3.
Figure 3.
Exon junction-specific mRNA expression of Abcb1a. Abundance of Abcb1a mRNAs with different lengths was assessed using assays covering the indicated exon junctions in female wild-type mice (n = 5), hABCB1 mice (n = 5), as well as in hABCB1 mice (n = 5) crossed to a Cre-deleter mouse strain (human ABCB1–/–). No differential expression between strains was found within each assay location. Data are mean ± standard deviation. Statistical significance was determined by 1-way ANOVA with Tukey's honest significant difference post-hoc test, significance level p < 0.05
Figure 4.
Figure 4.
Depiction of the humanized Abcb1a gene locus. Shown are the sequenced region (6089 bp, black) and the downstream adjacent mouse sequence not included in the sequencing (light grey). The human ABCB1 CDS was fused into the TSS, which left the untranslated base pairs TSS -1 to -6 of exon 2 behind (green). ABCB1 CDS has been introduced with its 3′UTR and additional 45 bp of human genomic downstream sequence. At the 3′ transition towards the mouse genomic sequence, a loxP site has been introduced which is flanked by altogether 99 bp of unknown origin. The same is true for the 5′ situated loxP site, which is flanked by additional 37 bp, respectively, and inserted into intron 1 of mouse Abcb1a. The picture is not proportional to the genomic arrangement.
Figure 5.
Figure 5.
Abcb1a promoter region analysis. Shown are the Abcb1a loci of wild-type and hABCB1 mice in comparison. ENCODE database searches revealed the indicated hotspots of open/accessible chromatin found in mouse brains using DNAse-seq, ATAC-seq, and ChIP-seq methods. The area of highest density of overlapping hotspots (orange/light orange) covers more than 200 bp indicating the importance of this region for Abcb1a gene expression.
Supplementary Figure 1.
Supplementary Figure 1.. Full exonic sequence of Abcb1a.
Capital letters indicate the coding sequence, lower case indicate untranslated bases. ATG start codons that could establish an open reading frame are indicated green. Exon junctions are indicated by an |. Pink letters indicate the central base of the probes of the TaqMan assays used (according manufactures information).
Supplementary Figure 1.
Supplementary Figure 1.. Full exonic sequence of Abcb1a.
Capital letters indicate the coding sequence, lower case indicate untranslated bases. ATG start codons that could establish an open reading frame are indicated green. Exon junctions are indicated by an |. Pink letters indicate the central base of the probes of the TaqMan assays used (according manufactures information).
Supplementary Figure 2.
Supplementary Figure 2.. Western blot analysis of ABCB1 expression
Western blot against ABCB1 confirmed PET imaging findings in hABCB1 animals and shows drastically reduced protein expression of any ABCB1 variant in hABCB1 as well as hABCB1–/– mice. Data are mean ± standard deviation. Statistical significance was determined by 1-way ANOVA with Tukey's Honest Significant Difference post-hoc test, significance level p<0.05, *** p<0.001.
Supplementary Figure 3.
Supplementary Figure 3.. In hABCB1 mice 266 bp of Abcb1a are deleted.
Alignment of hABCB1 sequence starting at the first base pair downstream of the loxP insertion site with Abcb1a starting at TSS.
Supplementary Figure 4.
Supplementary Figure 4.. Abcb1a exons 3 and 4 are unaltered.
Alignment of exon 3 (a) and exon 4 (b) as sequenced from hABCB1 mice with Abcb1a reference sequence (Gene ID: 18671). Bold letters indicate exons.

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