307 TRANSGENESIS MEDIATED BY INTRACYTOPLASMIC SPERM INJECTION (ICSI) ASSISTED BY CHEMICAL ACTIVATION IN DIFFERENT DOMESTIC SPECIES

Abstract

Intracytoplasmic sperm injection (ICSI)-mediated gene transfer has been described as a technique to obtain transgenic offspring in mice. However, this approach has had limited success in domestic animals due to poor embryo development after ICSI. A first experiment was designed to improve embryo development comparing ICSI-mediated gene transfer with or without chemical activation (CA) in the ovine species. In the second experiment, ICSI-mediated gene transfer assisted by CA was used in porcine, feline, equine, and bovine species. Maturation and culture were done by standard procedures. Semen was collected by artificial vagina in ovine and bovine species. In pigs, ejaculates were obtained using the gloved-hand method, and in feline and equine species, sperm were obtained from epididymides. Samples were frozen by standard means. Thawed spermatozoa were washed twice in Na citrate at 2.8% with 100 µm EDTA at 495g for 5 min and resuspended in Na citrate with 0.5 µg of pCX-EGFP/million spermatozoa for 5 min at 0°C. The pCX-EGFP plasmid contained the egfp gene expressed under chimerical CMV-IE-chicken β-actin promoter control. Sperm cells were immediately injected into the metaphase II oocyte and CA was induced by incubation in TALP-HEPES with 5 µm ionomycin for 4 min, cultured in TCM199 for 3 h, and transferred to a droplet of 1.9 mm 6-dimethylaminopurine (DMAP) for 3 h. During the in vitro culture, exposure to blue light (488 nm) was performed to determine the percentage of green embryos, mosaic expression, and earliest stage of egfp expression. Fluorescence in situ hybridization analysis was performed labeling pCX-EGFP plasmid by nick translation for use as a probe. Statistical analysis was done by chi square. In ovine species, development to blastocyst stage (0/88 v. 3/86; P > 0.05) and number of green embryos (24/88 v. 39/86; P < 0.05) were greater with CA. The egfp expression in ovine embryos assisted by CA began at the 2- (7/39), 4- (9/39), or 8-cell (23/39). However, the expression in ovine embryos without CA occurred only at the 8-cell stage (24/24) stage. In porcine, bovine, feline, and equine species, green embryos were detected at a high proportion (33/55, 10/44, 9/35, and 5/17, respectively), and the percentage of fluorescent blastocysts was 2.3, 2.9, and 9.1% for ovine, feline, and bovine species, respectively. The egfp expression in porcine and feline embryos started at the 2-cell stage (36 and 22%, respectively), whereas it began in bovine and equine embryos at the 4-cell stage (9 and 40% respectively). All species showed a high frequency of mosaic expression (range 60-85%), and the preliminary FISH analysis demonstrated a variable number of integration events in porcine embryos. To our knowledge, this is the first report of exogenous DNA expression in feline and equine embryos. These results suggest that the CA accelerates and increases the pCX-EGFP expression in ovine embryos in agreement with previous studies that have shown earlier expression of genes for parthenogenetic and cloning embryos, both assisted by CA. In conclusion, ICSI-mediated gene transfer assisted by CA can be used to obtain exogenous gene-expressing embryos in domestic species with potential scientific and commercial interests.