Expression of pluripotency related genes in TALEN mediated goat transgenic embryos

Abstract

Transcription activator-like effector nucleases (TALENs) are highly efficient genome-editing tools that facilitate precise site-directed modifications by inducing double-strand breaks in the host genome, enabling targeted gene knock-in or knock-out. The Rosa26 locus has been widely recognized as a safe harbor for gene integration across various species. This study aimed to assess the quantitative expression of pluripotency-related genes in transgenic goat embryos produced via hand-guided cloning, using goat fetal fibroblast cells with a GFP gene integrated at the Rosa26 locus via TALEN-mediated genome editing. Goat fetal fibroblast cells were co-transfected with a donor vector carrying the GFP gene and TALEN expression vectors. Transgenic goat embryos were generated using GFP-expressing transfected cells as nuclear donors for hand-guided cloning. The early-stage developmental rates of transgenic embryos were comparable to those of non-transgenic (control) embryos. Quantitative PCR (qPCR) analysis revealed no significant differences (P< 0.05) in the relative expression of pluripotency markers OCT4 (0.996±0.298 vs. 1.00) and NANOG (0.744±0.354 vs. 1.00) between transgenic and non-transgenic cloned embryos. These findings indicate that TALEN-mediated transfection did not impair the regenerative capacity of donor cells, allowing for normal embryonic development comparable to non-transgenic embryos. Thus, TALENs represent a promising tool for targeted gene insertion in the production of site-specific transgenic embryos.