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Unstable expression of transgene is associated with the methylation of CAG promoter in the offspring from the same litter of homozygous transgenic mice

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Abstract

Transgenic animals have been established for studying gene function, improving animals’ production traits, and providing organ models for the exploration of human diseases. However, the stability of inheritance and transgene expression in transgenic animals has gained extensive attention. The unstable expression of transgene through DNA methyltransferase (DNMT) targeting to the methylation of transgenic DNA such as CAG promoter and Egfp coding region in homozygous transgenic animals is still unknown. In the present study, the offspring from the same litter of homozygous transgenic mice carrying ubiquitously expressed enhanced green fluorescence protein driven by CMV early enhancer/chicken β-actin (CAG) promoter was observed to have unstable expression of transgene Egfp, quantitative PCR, western blot and bisulfite sequencing were conducted to quantify the expressional characteristics and methylation levels in various tissues. The correlation between transgene expression and methylation was analyzed. We have found that transgene expression is dependent on the methylation of CAG promoter, but not Egfp coding region. We have also characterized the correlation between the methylation of CAG promoter and DNMT, and found that only Dnmt3b expression is correlated with the methylation of CAG promoter. In conclusion, Dnmt3b-related methylation of CAG promoter can inhibit the transgene expression and may result in the unstable expression of transgene in the offspring from the same litter of homozygous transgenic mice.

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Acknowledgments

This work is supported by National Nature Science Foundation of China (31072032), National Transgenic Biology Program of China (2009ZX08008-006B and 2011ZX08008-003) and Foundation of Taishan Scholar of Shandong Province.

Conflicts of interest

All authors declare that there are no conflicts of interest.

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Authors and Affiliations

  1. College of Animal Science and Technology, Qingdao Agricultural University, Qingdao, 266109, Shan Dong, China

    Yang Zhou, Teng Zhang, Qin-Kai Zhang, Ying Jiang, Deng-Gao Xu, Min Zhang, Wei Shen & Qing-Jie Pan

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  1. Yang Zhou
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  3. Qin-Kai Zhang
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Correspondence to Qing-Jie Pan.

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11033_2014_3385_MOESM1_ESM.pptx

Supplementary Fig. 1. Identification of transgenic mouse genotype. PCR results for the offspring from parental female transgenic mice mated with wild-type mice were all positive. Similarly, PCR results for the offspring of parental male transgenic mice mated with wild-type mice were all positive. Supplementary Fig. 2. Gene structure analysis. The fragment with 891 bp was represented as the purple horizontal line. The fragment between targeted CAG promoter region and Egfp coding region was represented as the red horizontal line. The detailed sequence (−99 – +720 bp) was at the bottom. The black horizontal lines indicated the location of the primers. Supplementary Fig. 3. CpG island prediction of CAG promoter. The red horizontal line represented the input sequence. The blue vertical bars stood for the putative transcription factors that bind to specific loci. The red vertical lines represented the positions of 32CpG sites within the 276-bp fragment. The detailed CAG sequence (−352 – −76 bp) was under the CpG-pattern rich regions. The top sequence and bottom sequence correspond to the bisulfite sequencing and original sequence, respectively. The black horizontal lines indicated the location of primers. The black boxes indicated the distribution of CpG sites in the sequence. Supplementary Fig. 4. CpG island prediction of Egfp coding region. The red horizontal line represented the input sequence. The blue vertical bars stood for the putative transcription factors that bind to specific loci. The red vertical lines represented the positions of 15 CpG sites within the 191-bp fragment. The detailed Egfp sequence (+481 – +672 bp) was under the CpG-pattern rich regions. The top sequence and bottom sequence corresponded to the bisulfite sequencing and original sequence, respectively. The black horizontal lines revealed the location of primers. The black boxes presented the distribution of CpG sites in the sequence. (PPTX 189 kb)

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Zhou, Y., Zhang, T., Zhang, QK. et al. Unstable expression of transgene is associated with the methylation of CAG promoter in the offspring from the same litter of homozygous transgenic mice. Mol Biol Rep 41, 5177–5186 (2014). https://doi.org/10.1007/s11033-014-3385-1

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  • DOI: https://doi.org/10.1007/s11033-014-3385-1

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