81 MOUSE CLONING BY USING A LASER-ASSISTED ZONA OPENING AND ELECTRO-FUSION TECHNIQUE

Abstract

Mouse cloning can be performed by a direct microinjection of donor nuclei using a conventional or a piezo-actuated technique (Rybouchkin et al. 2002 Reproduction 124, 197–207; Wakayama et al. 1998 Nature 394, 369–374). However, a high percentage of lysed oocytes was observed during the pipette penetration of the cytoplasmic membrane through the zona pellucida. The aim of this experiment was to investigate the possibility of a combination of a laser-assisted zona opening and electro-fusion for mouse cloning. Mature oocytes were obtained from FSH-superovulated B6D2F1 female mice. Enucleation and transfer of donor cell were performed in HEPES-buffered CZB medium. Spindle-chromosome complexes (SCCs) together with first polar body were removed by blunt-end pipette via a small hole in the zona pellucida which was cut by a laser beam. An adult fibroblast cell was introduced into the perivitelline space and fused to the enucleated oocyte by using a single DC pulse of 1.5 kV cm^-1, 20 µs, in a fusion medium (Liu and Aoki 2003 Animal Sci. J. 75, 125–129). The fusion rate was checked 30 min later and only the fused oocytes were subjected to activation by 6 h culture in Ca²⁺-free CZB medium supplemented with 10 mM Sr²⁺ and 5 µg mL^-1 cytochalasin B. The oocytes which presented the pseudo-pronuclei were considered as the activated oocytes and were cultured in CZB medium at 37°C, 5% CO₂ in humidified atmosphere. The developmental rate was observed every 24 h for 4 days. The diploid parthenogenetically activated embryos serving as a control were obtained using the same activation protocol but without SCC removal. The percentages of survival after enucleation and after fusion were recorded. The formation of pseudo-pronuclei and the embryos developing to a particular stage were determined by chi-square analysis. The results show that most of the oocytes survived after enucleation (92.5%, 172/186) and the fusion rate was 71.9% (105/146). The formation of pseudo-pronuclei and the cleavage rate of cloned embryos was lower than in the control (87.6% (92/105) vs. 100% (90/90) and 69.6% (64/92) vs. 92.2% (83/90), respectively). The developmental rate to morula–blastocyst stage of cloned embryos was significantly lower than in the control [1.1% (1/92) vs. 44.4% (40/90); P < 0.05]. These results indicate that using laser-assisted zona opening and electro-fusion technique is practical for mouse cloning and provides an alternative method when injection of donor nuclei into the recipient oocytes using a conventional or a piezo-driven method is technically difficult.

This study was supported by grants from The National Research Council of Thailand and The Thailand Research Fund (Loyal Golden Julilee Ph.D. program).