Indian Journal of Science and Technology

Abstract

Objective: To produce bovine transgenic embryos by microinjection of a lentiviral vector that carries the eGFP gene as a marker in zygotes six hours after fertilization. Methods: 834 oocytes were matured and subjected to one of four treatments designed as follows: CC: Control: IVF with Cumulus-oocyte with (COCs), cultivated in CR2 medium supplemented with 10% FBS and incubated at 38.5°C in atmosphere of 95% humidity and 5% CO₂.CCM: Control culture medium: fertilized in vitro for six hours, cultured in medium SOF aluminum in pouches under the same conditions of CC. MC: Microinjection control: Fertilized under the same treatment conditions CCM. After six hours they were microinjected with TALP medium and cultured in sachets with the same conditions of CCM treatment. ML: Microinjected with the lentivirus: Fertilized in the same conditions of the CCM treatment. After six hours they were microinjected with the lentiviral vector carrying the eGFP transgene and cultured in sachets with the same treatment conditions CCM and MC. Findings: The cleavage rate found in CC was higher (p < 0.05) than that observed in the other treatments. The rates of blastocysts found between CC, CCM and MC did not differ significantly (p > 0.05) in them, but yes, with ML (p < 0.05). On average, 76.4% of the zygotes obtained in ML expressed the green fluorescent protein. Application/Improvements: The cult ure conditions used were suitable for CC, CCM and MC, microinjection with lentiviral vector has some influence on embryo development, it succeeded in obtaining transgenic zygotes.

Keywords: Green Fluorescent Protein (eGFP), Genetic Modification, Micromanipulation of Zygotes