ABSTRACT. Fluorescence expression by bovine embryos was examined after pronuclear microinjection with an enhanced green fluorescent protein (EGFP) cDNA under control of the chicken beta-actin promoter and cytomegalovirus enhancer, as a first step in evaluating the applicability of EGFP for non-invasive selection of transgenic bovine embryos. After injection, developmental competence of the embryos was reduced, and light was emitted in 11.9% of them (37/310) under a fluorescence microscope. Although 2.9% of the injected embryos developed to the fluorescent blastocysts (9/310), a majority of the fluorescent embryos showed mosaic expression including the negative blastomeres (26/37, 70.3%). These results suggest the feasibility of EGFP for in vitro selection of transgenic bovine embryos by fluorescence microscopy. However, the impaired development and high frequency of mosaicism were observed in these injected embryos.-KEY WORDS: bovine embryo, enhanced green fluorescent protein, marker.J. Vet. Med. Sci. 61 (7): [843][844][845][846][847] 1999 Briefly, cumulus-oocyte complexes (COCs) were aspirated from the follicles (2-7 mm in diameter) of cow ovaries collected at a local abattoir and cultured in maturation medium [25 mM Hepes TCM-199 with Eagle's salts (Gibco, Grand Island, NY) supplemented with 5% superovulated cow serum (SCS), 0.01 mg/ml folliclestimulating hormone (FSH, Denka, Kawasaki, Japan), 20 mM taurine (Wako, Osaka, Japan), and 50 µg/ml gentamicin (Sigma, St. Louis, MO)] at 38.5°C under 5% CO 2 in air. After 20-22 hr of culture, the COCs were fertilized in vitro with frozen-thawed sperm for 5 hr in Brackett and Olphant medium [3] containing 2.5 mM caffeine (Sigma), 3 mg/ml bovine serum albumin (BSA, fraction V; Sigma), 20 µg/ml heparin (Shimizu Pharmaceuticals, Shimizu, Japan) and 20 mM taurine, and then cultured in vitro in TCM-199 supplemented with 5% SCS, 5 µg/ml insulin (Sigma), 20 mM taurine, and 50 µg/ml gentamicin.After 17 hr of IVF, these presumptive zygotes were incubated with 300 IU/ml hyaluronidase (Sigma) for 20 min, and their adherent cumulus cells were removed by repeated pipetting. The denuded zygotes were centrifuged at 16,000 g for 20 min in the presence of 5 µg/ml cytochalasin B (Sigma) to visualize the pronuclei [9], and transferred to a 100 µl drop of Dulbecco's phosphate buffered saline without Ca 2+ and Mg 2+ [DPBS (-); Gibco] supplemented with 3 mg/ ml BSA, 5 µg/ml cytochalasin B, and 50 mg/ml gentamicin. After transfer, the pronuclei of the zygotes were microinjected with approximately 2 pl of the buffer solution (10 mM Tris-HCl, 0.2 mM EDTA, pH 7.5) containing 1.5 µg/ml EGFP cDNA fragment under control of the chicken beta-actin promoter and cytomegalovirus enhancer [11] using an injection capillary (0.5 µm in diameter) attached to a micromanipulator (Leitz, Wetzlar, Germany). These injected zygotes were cultured in vitro for an additional 8 days (until day 9; IVF = day 0) to examine their developmental competence and fluorescent expression.The green-light emission by the inject...
