CAG-GFP Lentivirus

Nonhuman primates (NHPs) are considered the most valuable model for the study of human transgenic (Tg) diseases, as NHPs more closely resemble human pathology compared to rodents. Previous studies have reported the generation of Tg NHPs that ubiquitously overexpress transgenes using various promoters. Here, researchers evaluated four putative ubiquitous promoters, namely, the cytomegalovirus (CMV) immediate early enhancer and chicken β-actin (CAG), elongation factor 1α (EF1α), ubiquitin C (UbC), and CMV, using an in vitro differentiation system of cynomolgus monkey embryonic stem cells (ESCs). While the EF1α promoter promoted Tg expression more than the other promoters in undifferentiated pluripotent ESCs, the CAG promoter was more effective in differentiated cells such as embryoid bodies and ESC-derived neurons. When both CAG and EF1α promoters were used to generate Tg monkeys expressing green fluorescent protein (GFP), the CAG promoter promoted GFP expression in skin and hematopoietic tissues more than EF1α-GFP Tg monkeys. Therefore, the CAG promoter appears to be the most suitable for ubiquitous and stable expression of transgenes in differentiated tissues of Tg cynomolgus monkeys and for establishing human disease models.

Because the CAG and EF1α promoters drove transgenic expression most strongly in differentiated and undifferentiated cells, respectively, lentiviral vectors carrying a GFP-encoding gene under the control of these promoters were constructed (Figure 1A) and injected into oocytes. Eight days after fertilization, the blastocysts infected with EF1α-GFP lentivirus showed stronger GFP fluorescence than did those treated with the CAG-GFP lentivirus (Figure 1B), consistent with the idea that the EF1α promoter drives stronger Tg expression than the CAG promoter in undifferentiated ESCs. After transfer to recipient foster mothers, three CAG-GFP Tg and three EF1α-GFP Tg offspring were obtained (Figure 1C). GFP fluorescence was observed on the facial skin of the CAG-GFP CE1894M, CE1993F and CE1984F Tg offspring, while GFP fluorescence was not detected on the facial skin of the EF1α-GFP Tg monkey (CE1881M), and GFP fluorescence was observed on the facial skin of the CE1886M and CE1887F. It is notable that GFP fluorescence was detected in a CAG-GFP Tg monkey carrying just one copy of the gene for GFP (CE1894M), whereas it was not detected in the EF1α-GFP Tg monkey carrying four copies of the transgenes (CE1881M) (Figure 1C). Thus, in ESC-derived differentiated cells, the CAG promoter drives transgene expression more strongly than the EF1α promoter, and the activity of the CAG promoter in the skin is stronger than that of the EF1α promoter overall (Figure 1).

Figure 1. Generation of GFP Tg cynomolgus monkeys. (Seita Y, et al., 2019)