Transcriptional activities of human elongation factor-1α and cytomegalovirus promoter in transgenic dogs generated by somatic cell nuclear transfer.

Kiyoung Eun,Yeon Woo Jeong,Seon-Ung Hwang,Min Gi Park,Hyunggee Kim,Yeon I K Jeong,Sang-Hwan Hyun,P Olof Olsson,Woo Suk Hwang,Nayoung Hong,Eun Ji Choi,Knut Stieger

doi:10.1371/journal.pone.0233784

Abstract

Recent advances in somatic cell nuclear transfer (SCNT) in canines facilitate the production of canine transgenic models. Owing to the importance of stable and strong promoter activity in transgenic animals, we tested human elongation factor 1α (hEF1α) and cytomegalovirus (CMV) promoter sequences in SCNT transgenic dogs. After transfection, transgenic donor fibroblasts with the hEF1α-enhanced green fluorescence protein (EGFP) transgene were successfully isolated using fluorescence-activated cell sorting (FACS). We obtained four puppies, after SCNT, and identified three puppies as being transgenic using PCR analysis. Unexpectedly, EGFP regulated by hEF1α promoter was not observed at the organismal and cellular levels in these transgenic dogs. EGFP expression was rescued by the inhibition of DNA methyltransferases, implying that the hEF1α promoter is silenced by DNA methylation. Next, donor cells with CMV-EGFP transgene were successfully established and SCNT was performed. Three puppies of six born puppies were confirmed to be transgenic. Unlike hEF1α-regulated EGFP, CMV-regulated EGFP was strongly detectable at both the organismal and cellular levels in all transgenic dogs, even after 19 months. In conclusion, our study suggests that the CMV promoter is more suitable, than the hEF1α promoter, for stable transgene expression in SCNT-derived transgenic canine model.

Highlights

Transgenic animal models are widely used in both basic research and preclinical studies
Production of somatic cell nuclear transfer (SCNT) transgenic dogs of enhanced green fluorescence protein (EGFP) gene controlled by human elongation factor 1α (hEF1α) and CMV promoter
We examined EGFP expression in fibroblasts isolated from each of the cloned puppies using fluorescence-activated cell sorting (FACS) and fluorescence microscopy, and found no EGFP expression in pups derived from K9-hEF1-EGFP, donor cells exhibited strong and stable EGFP expression before conducting SCNT (Fig 2D and 2E)

Summary

Introduction

Transgenic animal models are widely used in both basic research and preclinical studies. Over the past two decades, genetic engineering techniques such as zinc-finger nucleases-, Cre/loxP-, and CRISPR/Cas9-based site-specific genome editing methods, and gene silencing by RNA interference, have been developed and optimized for various human and animal systems. These technologies have resulted in a better platform for producing transgenic animals [2,3,4]. It is important to design a gene expression vector system capable of inducing the expression of the transgene and to demonstrate a clear in vivo phenotypic expression This requires the use of appropriate genetic elements, including promoters, introns, protein coding sequences, and polyadenylation signals [16]. We compared, the most widely used promoter sequence for considerably strong and stable transgene expression, human elongation factor 1α (hEF1α) and Cytomegalovirus (CMV) promoter, in SCNT transgenic dogs by detecting enhanced green fluorescence protein (EGFP)

Ethics statement

Evaluation of retrieved oocytes

Results

Discussion