Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seongbuk-gu, Seoul, Republic of Korea.
Institute of Animal Molecular Biotechnology, Korea University, Seongbuk-gu, Seoul, Republic of Korea.
PLoS One. 2020 Jun 3;15(6):e0233784. doi: 10.1371/journal.pone.0233784. eCollection 2020.
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.
近年来，犬体细胞核移植（SCNT）技术的进步促进了犬转基因模型的产生。由于在转基因动物中稳定且强的启动子活性非常重要，我们在 SCNT 转基因犬中测试了人延伸因子 1α（hEF1α）和巨细胞病毒（CMV）启动子序列。转染后，使用荧光激活细胞分选（FACS）成功分离出携带 hEF1α-增强型绿色荧光蛋白（EGFP）转基因的转基因供体细胞。我们通过 SCNT 获得了四只小狗，并通过 PCR 分析鉴定了其中三只小狗为转基因。出乎意料的是，在这些转基因犬中，未观察到由 hEF1α 启动子调控的 EGFP 在机体和细胞水平上的表达。通过抑制 DNA 甲基转移酶，EGFP 的表达得到了挽救，这表明 hEF1α 启动子被 DNA 甲基化沉默。接下来，成功建立了携带 CMV-EGFP 转基因的供体细胞，并进行了 SCNT。在六只出生的小狗中，有三只被确认为转基因。与 hEF1α 调控的 EGFP 不同，CMV 调控的 EGFP 在所有转基因犬的机体和细胞水平上都能强烈检测到，即使在 19 个月后也是如此。总之，我们的研究表明，CMV 启动子比 hEF1α 启动子更适合在 SCNT 衍生的转基因犬模型中稳定表达转基因。
