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

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