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A preliminary exploration on DNA methylation of transgene across generations in transgenic rats.

Li Q, Xu W, Cui Y, Ma L, Richards J, Li W, Ma Y, Fu G, Bythwood T, Wang Y, Li X, Song Q - Sci Rep (2015)

Bottom Line: We performed bisulfite sequencing on 23 CpG dinucleotides on the transgene across three generations in two tissues.We observed that the transgene was heavily methylated in the liver (87.53%) from the founder generation, whereas its methylation rate was much lower in the kidney (70.47%).Spearman correlation analysis showed that there was a strong correlation on the methylation status between different generations in the same tissue, which was observed in both liver and kidney, and among all individuals in this pedigree.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Obstetrics and Gynecology, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, China [2] Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta, Georgia, USA.

ABSTRACT
Epigenetic heritability is an important issue in the field of genetics and also in the development of many human diseases. In this study, we created a transgenic rat model and investigated the transgenerational methylation patterns in these animals. The transgene DNA fragment was unmethylated before it was injected into the pronucleus, so it is a good model to study the inheritance of DNA methylation patterns. We performed bisulfite sequencing on 23 CpG dinucleotides on the transgene across three generations in two tissues. We observed that the transgene was heavily methylated in the liver (87.53%) from the founder generation, whereas its methylation rate was much lower in the kidney (70.47%). Spearman correlation analysis showed that there was a strong correlation on the methylation status between different generations in the same tissue, which was observed in both liver and kidney, and among all individuals in this pedigree. This study provided some evidence that DNA methylation patterns acquired in the founder animal can be passed to the offspring.

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Related in: MedlinePlus

The three-generation pedigree of rats in this study.SD, Sprague-Dawley. Squares indicate male rats; circles indicate female rats. Rat ID was labeled in the squares or circles.
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f3: The three-generation pedigree of rats in this study.SD, Sprague-Dawley. Squares indicate male rats; circles indicate female rats. Rat ID was labeled in the squares or circles.

Mentions: The transgene contained human CRP gene (21 bp fragment before the transcription starting site, the exons and intron, and 1.2 kb of 3′-flanking region), and mouse albumin promoter (from +22 to −305 bp) and enhancer (from −12.171 kb to −9.469 kb)14. Purified DNA was microinjected into fertilized eggs of Sprague-Dawley (SD) rats (Charles River Laboratory, Wilmington, MA). Pronuclear microinjection was performed at the University of Michigan Transgenic Animal Model Core Facility. Transgenic rats were identified by PCR with transgene-specific primers (Forward 5′-ACATACGCAAGGGATTTAGTC-3′; Reverse 5′-AACAGCTTCTCCATGGTCAC-3′) using genomic DNA samples obtained from tail biopsies. Founder rats were bred with non-transgenic SD rats to establish transgenic lines. Animals were housed in the Center for Laboratory Animal Resources of Morehouse School of Medicine. Transgenic CRP rats were given water ad libitum and a standard rat chow diet (Laboratory Rodent Diet 5001, LabDiet, USA). All animal experiments were performed with the approval of the Animal Care Committee of Morehouse School of Medicine, and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Five transgenic rats at 15–16 weeks old in three generations were chosen in this study (Figure 3). Among these 5 rats, Rat-1 was a founder rat, Rats 2–4 were the second generation of heterozygous offspring, and Rat-5 was the third generation heterozygous offspring.


A preliminary exploration on DNA methylation of transgene across generations in transgenic rats.

Li Q, Xu W, Cui Y, Ma L, Richards J, Li W, Ma Y, Fu G, Bythwood T, Wang Y, Li X, Song Q - Sci Rep (2015)

The three-generation pedigree of rats in this study.SD, Sprague-Dawley. Squares indicate male rats; circles indicate female rats. Rat ID was labeled in the squares or circles.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321119&req=5

f3: The three-generation pedigree of rats in this study.SD, Sprague-Dawley. Squares indicate male rats; circles indicate female rats. Rat ID was labeled in the squares or circles.
Mentions: The transgene contained human CRP gene (21 bp fragment before the transcription starting site, the exons and intron, and 1.2 kb of 3′-flanking region), and mouse albumin promoter (from +22 to −305 bp) and enhancer (from −12.171 kb to −9.469 kb)14. Purified DNA was microinjected into fertilized eggs of Sprague-Dawley (SD) rats (Charles River Laboratory, Wilmington, MA). Pronuclear microinjection was performed at the University of Michigan Transgenic Animal Model Core Facility. Transgenic rats were identified by PCR with transgene-specific primers (Forward 5′-ACATACGCAAGGGATTTAGTC-3′; Reverse 5′-AACAGCTTCTCCATGGTCAC-3′) using genomic DNA samples obtained from tail biopsies. Founder rats were bred with non-transgenic SD rats to establish transgenic lines. Animals were housed in the Center for Laboratory Animal Resources of Morehouse School of Medicine. Transgenic CRP rats were given water ad libitum and a standard rat chow diet (Laboratory Rodent Diet 5001, LabDiet, USA). All animal experiments were performed with the approval of the Animal Care Committee of Morehouse School of Medicine, and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Five transgenic rats at 15–16 weeks old in three generations were chosen in this study (Figure 3). Among these 5 rats, Rat-1 was a founder rat, Rats 2–4 were the second generation of heterozygous offspring, and Rat-5 was the third generation heterozygous offspring.

Bottom Line: We performed bisulfite sequencing on 23 CpG dinucleotides on the transgene across three generations in two tissues.We observed that the transgene was heavily methylated in the liver (87.53%) from the founder generation, whereas its methylation rate was much lower in the kidney (70.47%).Spearman correlation analysis showed that there was a strong correlation on the methylation status between different generations in the same tissue, which was observed in both liver and kidney, and among all individuals in this pedigree.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Obstetrics and Gynecology, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, China [2] Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta, Georgia, USA.

ABSTRACT
Epigenetic heritability is an important issue in the field of genetics and also in the development of many human diseases. In this study, we created a transgenic rat model and investigated the transgenerational methylation patterns in these animals. The transgene DNA fragment was unmethylated before it was injected into the pronucleus, so it is a good model to study the inheritance of DNA methylation patterns. We performed bisulfite sequencing on 23 CpG dinucleotides on the transgene across three generations in two tissues. We observed that the transgene was heavily methylated in the liver (87.53%) from the founder generation, whereas its methylation rate was much lower in the kidney (70.47%). Spearman correlation analysis showed that there was a strong correlation on the methylation status between different generations in the same tissue, which was observed in both liver and kidney, and among all individuals in this pedigree. This study provided some evidence that DNA methylation patterns acquired in the founder animal can be passed to the offspring.

Show MeSH
Related in: MedlinePlus