DNMT 1 maintains hypermethylation of CAG promoter specific region and prevents expression of exogenous gene in fat-1 transgenic sheep

Abstract

Methylation is an important issue in gene expression regulation and also in the fields of genetics and reproduction. In this study, we created fat-1 transgenic sheep, investigated the fine-mapping and the modulatory mechanisms of promoter methylation. Sheep fetal fibroblasts were transfected by pCAG-fat1-IRES-EGFP. Monoclonal cell line was screened as nuclear donor and carried out nuclear transfer (441 transgenic cloned embryos, 52 synchronism recipient sheep). Six offsprings were obtained. Expressions of exogenous genes fat-1 and EGFP were detectable in 10 examined tissues and upregulated omega-3 fatty acid content. Interestingly, more or less EGFP negative cells were detectable in the positive transgenic fetal skin cells. EGFP negative and positive cells were sorted by flow cytometry, and their methylation status in the whole promoter region (1701 nt) were investigated by bisulphate sequencing. The fine-mapping of methylation in CAG promoter were proposed. The results suggested that exogenous gene expression was determined by the methylation status from 721–1346 nt and modulated by methylation levels at 101, 108 and 115 nt sites in CAG promoter. To clarify the regulatory mechanism of methylation, examination of four DNA methyltransferases (DNMTs) demonstrated that hypermethylation of CAG promoter is mainly maintained by DNMT 1 in EGFP negative cells. Furthermore, investigation of the cell surface antigen CD34, CD45 and CD166 indicated that EGFP positive and negative cells belong to different types. The present study systematically clarified methylation status of CAG promoter in transgenic sheep and regulatory mechanism, which will provide research strategies for gene expression regulation in transgenic animals.

Comparative Transcriptome Analysis Provides Insights into the Polyunsaturated Fatty Acid Synthesis Regulation of Fat-1 Transgenic Sheep

“…The introduction of the Fat-1 gene in mouse, pig, sheep, cattle, and cow resulted in high levels of n-3 PUFAs and significantly lower n-6/n-3 PUFA ratios in their tissues [10][11][12]23,46,47]. In our study, the same results were obtained by comparing n-6 and n-3 PUFA contents between 100-day transgenic fetuses and wild type fetuses.…”

“…However, the transgenic fetuses possessed both Fat-1 expressing and silencing cells in their tissues. Our previous study indicated that the hypermethylation status in the specific CAG promoter region may be responsible for preventing Fat-1 expression in transgenic-silencing cells [23]. In the current study, the skin cells with or without Fat-1 expression were further compared at the transcriptome level.…”

“…Sheep that produce mutton with high n-3 PUFAs contents may benefit people's health and have high economic values. In the year 2012, our team produced Fat-1 transgenic sheep with sheep fetal fibroblasts transfected with the pCMV-fat-1-IREs-EGFP plasmid and pCAG-fat-1-IREs-EGFP plasmid as donor cells [22,23]. In the current study, in order to reveal the expression status of the exogenous Fat-1 gene and how it affects PUFA synthesis in transgenic fetuses and lambs, we collected tissue samples from abnormal and normal transgenic fetuses on the 100th gestational day, postnatal lambs, as well as transgenic silencing and expressing skin cells, to conduct transcriptome sequencing.…”

Transgenic mice as models to study the influence of polyunsaturated fatty acids on metabolism

“…Danio rerio fat1 oraz fat1/fat2 [37] szczur fat-1 [38] owca fat-1 [39] świnia hfat-1 [40] sFat-1 [41] krowa fat-1 [42] skrzyżowanie ze sobą dwóch heterozygotycznych myszy: fat-1 i fat-2 otrzymano myszy fat-1/fat-2 zwane myszami "Omega". Dzięki temu możliwym stała się synteza kwasów omega-6 i omega-3 przez ssaki z innych niż WNKT składników diety tzn.…”

Stillbirth risk on fat-1 transgenic foetus of sheep caused by deregulated DNA methylation of imprinted genes

“…Transgene has been the considerable controversy all around the world. In terms of fat-1 transgenic animals, the expression of exogenous gene fat-1 can be detected in tissues and upregulates omega-3 polyunsaturated fatty acid (n-3 PUFA) (Yang et al 2017). N-3 PUFAs, as essential fatty acids, cannot be synthesized in mammals (van West and Maes 2003).…”