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Sexually Dimorphic Expression of eGFP Transgene in the Akr1A1 Locus of Mouse Liver Regulated by Sex Hormone-Related Epigenetic Remodeling.

Lai CW, Chen HL, Tsai TC, Chu TW, Yang SH, Chong KY, Chen CM - Sci Rep (2016)

Bottom Line: However, the mechanism of sexually dimorphic expression is still not fully understood.In this study, a pCAG-eGFP transgenic mouse strain with a specific transgene integration site in the Akr1A1 locus presented male-biased EGFP expression in the liver, and the expression was activated by testosterone during puberty.The integration of the pCAG-eGFP transgene altered the epigenetic regulation of the adjacent chromatin, including increased binding of STAT5b, a sexually dimorphic expression regulator, and the transformation of DNA methylation from hypermethylation into male-biased hypomethylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, and Agricultural Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan.

ABSTRACT
Sexually dimorphic gene expression is commonly found in the liver, and many of these genes are linked to different incidences of liver diseases between sexes. However, the mechanism of sexually dimorphic expression is still not fully understood. In this study, a pCAG-eGFP transgenic mouse strain with a specific transgene integration site in the Akr1A1 locus presented male-biased EGFP expression in the liver, and the expression was activated by testosterone during puberty. The integration of the pCAG-eGFP transgene altered the epigenetic regulation of the adjacent chromatin, including increased binding of STAT5b, a sexually dimorphic expression regulator, and the transformation of DNA methylation from hypermethylation into male-biased hypomethylation. Through this de novo sexually dimorphic expression of the transgene, the Akr1A1(eGFP) mouse provides a useful model to study the mechanisms and the dynamic changes of sexually dimorphic gene expression during either development or pathogenesis of the liver.

No MeSH data available.


Related in: MedlinePlus

The integration of pCAG-eGFP in the Tg mouse line and mRNA expression of eGFP and the adjacent genes.(A) The proportional map of the pCAG-eGFP transgene integration position at the D1 band of mouse chromosome 4. There are 34 kb between exon 1 of Akr1A1 and the Prdx1 gene in WT mice, and the integration of the pCAG-eGFP transgene caused a ~30-kb genomic deletion, which includes exons 1–5 of the Akr1A1 gene and the non-coding region between the Akr1A1 and Prdx1 genes. The red and black boxes represent the exons of Akr1A1 and Prdx1 genes, respectively. The blue and green boxes represent the CAG promoter and eGFP of the transgene, respectively. ATG and TAG represent the start and stop codons of the Akr1A1 gene, respectively. P1, P2 and P3 represent the oligonucleotide primers that were used for the mouse genotyping by PCR. Scale bar: 1 kb. (B) The genotyping of the pCAG-eGFP mouse line by PCR. The 629 bp and 294 bp amplicons represent the transgenic allele and non-transgenic allele, respectively. (C) IHC of the AKR1A1 protein that was expressed in WT but not in Akr1A1eGFP/eGFP mouse liver. Scale bar: 400 μm. (D–F) mRNA expression of Akr1A1, eGFP and Prdx1 in the Akr1A1eGFP/+, Akr1A1eGFP/eGFP and WT mouse livers. The bars show the mean ± s.e.m. of five animals per group (n = 5); data were analyzed by a one-way ANOVA with post hoc comparisons using Duncan’s new multiple range test; **represents significant differences (P < 0.01).
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f2: The integration of pCAG-eGFP in the Tg mouse line and mRNA expression of eGFP and the adjacent genes.(A) The proportional map of the pCAG-eGFP transgene integration position at the D1 band of mouse chromosome 4. There are 34 kb between exon 1 of Akr1A1 and the Prdx1 gene in WT mice, and the integration of the pCAG-eGFP transgene caused a ~30-kb genomic deletion, which includes exons 1–5 of the Akr1A1 gene and the non-coding region between the Akr1A1 and Prdx1 genes. The red and black boxes represent the exons of Akr1A1 and Prdx1 genes, respectively. The blue and green boxes represent the CAG promoter and eGFP of the transgene, respectively. ATG and TAG represent the start and stop codons of the Akr1A1 gene, respectively. P1, P2 and P3 represent the oligonucleotide primers that were used for the mouse genotyping by PCR. Scale bar: 1 kb. (B) The genotyping of the pCAG-eGFP mouse line by PCR. The 629 bp and 294 bp amplicons represent the transgenic allele and non-transgenic allele, respectively. (C) IHC of the AKR1A1 protein that was expressed in WT but not in Akr1A1eGFP/eGFP mouse liver. Scale bar: 400 μm. (D–F) mRNA expression of Akr1A1, eGFP and Prdx1 in the Akr1A1eGFP/+, Akr1A1eGFP/eGFP and WT mouse livers. The bars show the mean ± s.e.m. of five animals per group (n = 5); data were analyzed by a one-way ANOVA with post hoc comparisons using Duncan’s new multiple range test; **represents significant differences (P < 0.01).

Mentions: The integration of the pCAG-eGFP transgene in this Tg mouse line was revealed by chromosome fluorescence in situ hybridization (FISH) and compatible ends ligation inverse PCR (CELI-PCR). The results showed only one transgene integration site (Fig. 1D), which was located in the region of the Akr1A1 gene locus that is on the D1 band of mouse chromosome 4 (Fig. 2A). The integration of the transgene caused an approximately 30 kb genomic DNA deletion, including exons 1–5 of the Akr1A1 gene and the non-coding region between the Akr1A1 and Prdx1 genes, which resulted in the knockout of the Akr1A1 gene and the decreased distance between the transgene and the Prdx1 gene, which are 10 kb apart (Fig. 2A–D). According to the nomenclature guidelines of the International Committee for Standardized Genetic Nomenclature for Mice, the Tg mice are denoted Akr1A1Tg(CAG-eGFP)Cmc. However, to simplify the nomenclature, the heterozygous (Akr1A1Tg (CAG-eGFP)Cmc/+) and homozygous (Akr1A1Tg (CAG-eGFP)Cmc/Tg (CAG-eGFP)Cmc) Tg mice are indicated by Akr1A1eGFP/+ and Akr1A1eGFP/eGFP, respectively, in the following experiments.


Sexually Dimorphic Expression of eGFP Transgene in the Akr1A1 Locus of Mouse Liver Regulated by Sex Hormone-Related Epigenetic Remodeling.

Lai CW, Chen HL, Tsai TC, Chu TW, Yang SH, Chong KY, Chen CM - Sci Rep (2016)

The integration of pCAG-eGFP in the Tg mouse line and mRNA expression of eGFP and the adjacent genes.(A) The proportional map of the pCAG-eGFP transgene integration position at the D1 band of mouse chromosome 4. There are 34 kb between exon 1 of Akr1A1 and the Prdx1 gene in WT mice, and the integration of the pCAG-eGFP transgene caused a ~30-kb genomic deletion, which includes exons 1–5 of the Akr1A1 gene and the non-coding region between the Akr1A1 and Prdx1 genes. The red and black boxes represent the exons of Akr1A1 and Prdx1 genes, respectively. The blue and green boxes represent the CAG promoter and eGFP of the transgene, respectively. ATG and TAG represent the start and stop codons of the Akr1A1 gene, respectively. P1, P2 and P3 represent the oligonucleotide primers that were used for the mouse genotyping by PCR. Scale bar: 1 kb. (B) The genotyping of the pCAG-eGFP mouse line by PCR. The 629 bp and 294 bp amplicons represent the transgenic allele and non-transgenic allele, respectively. (C) IHC of the AKR1A1 protein that was expressed in WT but not in Akr1A1eGFP/eGFP mouse liver. Scale bar: 400 μm. (D–F) mRNA expression of Akr1A1, eGFP and Prdx1 in the Akr1A1eGFP/+, Akr1A1eGFP/eGFP and WT mouse livers. The bars show the mean ± s.e.m. of five animals per group (n = 5); data were analyzed by a one-way ANOVA with post hoc comparisons using Duncan’s new multiple range test; **represents significant differences (P < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834580&req=5

f2: The integration of pCAG-eGFP in the Tg mouse line and mRNA expression of eGFP and the adjacent genes.(A) The proportional map of the pCAG-eGFP transgene integration position at the D1 band of mouse chromosome 4. There are 34 kb between exon 1 of Akr1A1 and the Prdx1 gene in WT mice, and the integration of the pCAG-eGFP transgene caused a ~30-kb genomic deletion, which includes exons 1–5 of the Akr1A1 gene and the non-coding region between the Akr1A1 and Prdx1 genes. The red and black boxes represent the exons of Akr1A1 and Prdx1 genes, respectively. The blue and green boxes represent the CAG promoter and eGFP of the transgene, respectively. ATG and TAG represent the start and stop codons of the Akr1A1 gene, respectively. P1, P2 and P3 represent the oligonucleotide primers that were used for the mouse genotyping by PCR. Scale bar: 1 kb. (B) The genotyping of the pCAG-eGFP mouse line by PCR. The 629 bp and 294 bp amplicons represent the transgenic allele and non-transgenic allele, respectively. (C) IHC of the AKR1A1 protein that was expressed in WT but not in Akr1A1eGFP/eGFP mouse liver. Scale bar: 400 μm. (D–F) mRNA expression of Akr1A1, eGFP and Prdx1 in the Akr1A1eGFP/+, Akr1A1eGFP/eGFP and WT mouse livers. The bars show the mean ± s.e.m. of five animals per group (n = 5); data were analyzed by a one-way ANOVA with post hoc comparisons using Duncan’s new multiple range test; **represents significant differences (P < 0.01).
Mentions: The integration of the pCAG-eGFP transgene in this Tg mouse line was revealed by chromosome fluorescence in situ hybridization (FISH) and compatible ends ligation inverse PCR (CELI-PCR). The results showed only one transgene integration site (Fig. 1D), which was located in the region of the Akr1A1 gene locus that is on the D1 band of mouse chromosome 4 (Fig. 2A). The integration of the transgene caused an approximately 30 kb genomic DNA deletion, including exons 1–5 of the Akr1A1 gene and the non-coding region between the Akr1A1 and Prdx1 genes, which resulted in the knockout of the Akr1A1 gene and the decreased distance between the transgene and the Prdx1 gene, which are 10 kb apart (Fig. 2A–D). According to the nomenclature guidelines of the International Committee for Standardized Genetic Nomenclature for Mice, the Tg mice are denoted Akr1A1Tg(CAG-eGFP)Cmc. However, to simplify the nomenclature, the heterozygous (Akr1A1Tg (CAG-eGFP)Cmc/+) and homozygous (Akr1A1Tg (CAG-eGFP)Cmc/Tg (CAG-eGFP)Cmc) Tg mice are indicated by Akr1A1eGFP/+ and Akr1A1eGFP/eGFP, respectively, in the following experiments.

Bottom Line: However, the mechanism of sexually dimorphic expression is still not fully understood.In this study, a pCAG-eGFP transgenic mouse strain with a specific transgene integration site in the Akr1A1 locus presented male-biased EGFP expression in the liver, and the expression was activated by testosterone during puberty.The integration of the pCAG-eGFP transgene altered the epigenetic regulation of the adjacent chromatin, including increased binding of STAT5b, a sexually dimorphic expression regulator, and the transformation of DNA methylation from hypermethylation into male-biased hypomethylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, and Agricultural Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan.

ABSTRACT
Sexually dimorphic gene expression is commonly found in the liver, and many of these genes are linked to different incidences of liver diseases between sexes. However, the mechanism of sexually dimorphic expression is still not fully understood. In this study, a pCAG-eGFP transgenic mouse strain with a specific transgene integration site in the Akr1A1 locus presented male-biased EGFP expression in the liver, and the expression was activated by testosterone during puberty. The integration of the pCAG-eGFP transgene altered the epigenetic regulation of the adjacent chromatin, including increased binding of STAT5b, a sexually dimorphic expression regulator, and the transformation of DNA methylation from hypermethylation into male-biased hypomethylation. Through this de novo sexually dimorphic expression of the transgene, the Akr1A1(eGFP) mouse provides a useful model to study the mechanisms and the dynamic changes of sexually dimorphic gene expression during either development or pathogenesis of the liver.

No MeSH data available.


Related in: MedlinePlus