EXPRESSION OF GFP GENE DURING PREIMPLATATION DEVELOPMENT OF TRANSGENIC CLONED BOVINE EMBRYO

Abstract

This study aimed to investigate the expression of the green fluorescent protein (GFP) gene during the preimplantation development of transgenic cloned bovine embryos. Oocyte-cumulus-granulosa cell complexes (OCGs) were collected from bovine ovaries for in-vitro maturation, and fibroblasts of Holstein cow were cultured for pMax-GFP gene transfection and nuclear transfer. Somatic cell nuclear transfer was performed to generate transgenic cloned bovine embryos by the advance of micromanipulation system to collect the transfected cells. The expression of GFP proteins were observed throughout transfected fibroblasts culturing and transgenic cloned bovine embryos preimplantation development by using UV light. The results showed that the expression of the GFP gene in fibroblasts decreased over time. At day 3, 42.3% of the cells expressed the GFP gene, while at day 6, the percentage decreased to 15.6%. By day 10, after cell passage, the percentage further decreased to 8.2%. Besides that, the GFP gene was expressed in the 2-cell embryo and blastocyst stages of preimplantation development of transgenic cloned bovine embryos. The GFP expression was comparatively low during the 2-cell embryo stage. However, at blastocyst stage the level of GFP expression were relatively high. The non-transfected group had a higher percentage of embryos developing to the blastocyst stage at 29.6% compared to the transfected group at 21.05%. Additionally, the cell number of the blastocyst was higher in the non-transfected group (102 cells) compared to the transfected group (54 cells). These observations suggest that the foreign gene could be expressed during the developmental progress of transgenic cloned bovine embryos derived from transfected donor cells. The study highlights the potential utility genome editing technology and reproductive biotechnology for generating transgenic cloned bovine embryos, which could be particularly apply for further production of genetically modified livestock with desirable traits. Overall, this study provides a better understanding of the foreign gene expression in transfected cow fibroblast and during early embryonic development that could contribute to the development of more efficient methods for producing transgenic cloned production