Production of IVF transgene-expressing bovine embryos using a novel strategy based on cell cycle inhibitors.

摘要: The objective was to evaluate the effects of cell cycle inhibitors (6-dimethylaminopurine [DMAP], and dehydroleukodine [DhL]) on transgene expression efficiency mosaic patterns IVF bovine zygotes cytoplasmically injected with oolema vesicles coincubated transgene. DNA damage induced by or measured detection phosphorylated histone H2AX foci presence (marker double-stranded breaks). Cloning egfp blastomeres included determine continuity after additional rounds cellular division. pCX-EGFP [enhanced green fluorescent protein gene (EGFP) under chimeric cytomegalovirus IE-chicken-β-actin enhancer promoter control] plasmid (50 ng/μL) alone (linear circular exogenous DNA, leDNA ceDNA, respectively) associated ooplasmic (leDNA-v ceDNA-v). 2 mm DMAP 1 μm DhL for 6 h (from 15 21 post IVF) evaluated groups vesicles. increased (P < 0.05) homogenous relative (21%, 18%, 11% leDNA-v + DMAP, leDNA-v, leDNA, also area. Expression higher linear than pCX-EGFP, blastocyst rates were (95%, 77%, 84%, 52% DhL, respectively). Moreover, tended improve blastocysts both transgenes. Based in situ hybridization (FISH) analysis, there evidence integration embryos. Finally, clones derived from had highest (96%, 65%, 65% Transgenesis cytoplasmic injection is a promising alternative transgenic animal production.