411 EFFECTIVE PRODUCTION OF ICSI-MEDIATED TRANSGENIC MICE: INTENSITY OF GFP EXPRESSION AS A MARKER GENE AND INCIDENCE OF APOPTOSIS IN BLASTOCYSTS

Abstract

Transgenic animals provide a powerful model to explore the regulation of gene expression, and to study gene functionality. The objective of this study was to improve the production efficiency of transgenic mice in ICSI-mediated transgenesis. We examined the relation between the intensity of green fluorescent protein (GFP) expression as a marker gene in blastocysts and the productivity of green mice after embryo transfer, and the incidence of apoptosis in blastocysts with GFP expression. Spermatozoa were processed fresh (FR) and with 2 methods that cause membrane disruption: freeze–thawing (FT), or freeze–thawing followed by 0.5% Triton X-100 for 0.5 min (TX). The spermatozoa were co-incubated with 4.0 ng µL^-1 of pCX-EGFP DNA fragments (3.5 kb) for 1 min on ice and injected into oocytes of B6D2F1 mice. The injected oocytes were cultured in KSOMaa for 96 h. Blastocysts were classified into 3 categories depending on the intensity of GFP expression (strong, moderate, and mosaic/negative) under fluorescence microscopy. The number of apoptotic cells in the blastocysts was determined by the TUNEL method using an in situ cell death detection kit (Roche Diagnostics, Mannheim, Germany). Data were analyzed by the chi-square test in all experiments. Blastocyst rates for FR, FT, and TX were 77% (27/35), 67% (18/27), and 69% (31/45), respectively. The proportion of blastocysts expressing GFP for FR, FT, and TX were 0% (0/27), 22% (4/18), and 52% (16/31), respectively. The percentage of apoptotic cells (the number of apoptotic cells/total cells) in blastocysts with strong GFP expression (7.3 ± 0.5%; 4.9 ± 0.4/68 ± 3) was significantly higher (P < 0.05) than in those with moderate (2.5 ± 0.3%; 2.2 ± 0.2/88 ± 3) or mosaic/negative (1.5 ± 0.2%; 1.4 ± 0.2/91 ± 2) GFP expression. After embryo transfer, the rates of implantation, fetal development, and green fetus production were significantly higher (P < 0.05) in blastocysts with moderate GFP expression (52%, 16/31; 19%, 6/312 and 16%, 5/31, respectively) than in those with strong GFP expression (12%, 4/33; 6%, 2/33; 3%, 1/33). These results suggest that a high amount of GFP expression in blastocyst is detrimental to embryo development and survival.