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Construction and Analysis of an Adipose Tissue-Specific and Methylation-Sensitive Promoter of Leptin Gene

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Abstract

DNA methylation plays a very important role in the regulation of gene expression. Under general situations, methylation in a gene promoter region is frequently accompanied by transcriptional suppression, and those genes that are highly methylated display the phenomenon of low expression. In contrast, those genes whose methylation level is low display the phenomenon of active expression. In this study, we conducted DNA methylation analysis on the CpG sites within the promoter regions of five adipose tissue-specific transcriptional factors—Adiponectin, Chemerin, Leptin, Smaf-1, and Vaspin—and examined their messenger RNA (mRNA) expression levels in different mouse tissues. We also performed analyses on the correlation between the DNA methylation levels of these genes and their mRNA expression levels in these tissues. The correlation coefficient for Leptin was the highest, and it displayed a high expression in an adipose tissue-specific manner. Thus, we cloned the regulatory region of Leptin gene and incorporated its promoter into the eukaryotic expression vector pEGFP-N1 and constructed a recombinant plasmid named pEGFP-N1-(p-Lep). This recombinant plasmid was first verified by DNA sequencing and then transfected into mouse pre-adipocytes via electroporation. Measurement of the activity of luciferase (reporter) indicated that p-Lep was capable of driving the expression of the reporter gene. This study has paved a solid basis for subsequent studies on generating transgenic animals.

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Acknowledgments

This work is supported by the National Nature Science Foundation of China (31072032), National Transgenic Biology Program of China (2011ZX08008-003), and Taishan Scholar Construction Foundation, P.R. China (631114).

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    Authors and Affiliations

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

      Qinkai Zhang, Denggao Xu, Min Zhang, Xiao Dong, Huansheng Dong & Qingjie Pan

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    1. Qinkai Zhang
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    2. Denggao Xu
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    3. Min Zhang
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    4. Xiao Dong
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    5. Huansheng Dong
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    6. Qingjie Pan
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    Correspondence to Huansheng Dong or Qingjie Pan.

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    Qinkai Zhang and Denggao Xu contributed equally to this work.

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    Zhang, Q., Xu, D., Zhang, M. et al. Construction and Analysis of an Adipose Tissue-Specific and Methylation-Sensitive Promoter of Leptin Gene. Appl Biochem Biotechnol 180, 1213–1226 (2016). https://doi.org/10.1007/s12010-016-2162-0

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    • DOI: https://doi.org/10.1007/s12010-016-2162-0

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