3 MEGANUCLEASE TRANSGENESIS IN IVF AND CLONED BOVINE PREIMPLANTATORY EMBRYOS

Abstract

Techniques available for production of transgenic animals remain inefficient. Recently an effective transgenesis system using I-SceI meganuclease (MegaN) has been described in Xenopus. It consists of MegaN incubation with a pIS-plasmid carrying I-SceI recognition sites-, followed by injection into fertilized egg. Here, we evaluated the MegaN transgenesis system combined with new transgenesis approaches developed by our group. We compared (1) three pIS concentrations incubated or not with MegaN; (2) injection of oocytes at germinal vesicle stage (GV) or of presumptive zygotes post IVF; (3) cloning using donor cells briefly incubated with MegaN or with pIS alone. Conditions for MegaN digestion were 0.5 UI I-SceI + 1x I-SceI buffer +pIS, at 37°C for 45 min. Two pIS were tested. For #1, IS- PaxGFP at 15, 25 and 50 ng μL^–1 (15+, 25+ and 50+ for transgene digested by MegaN and 15-, 25- and 50- for transgene not digested). For #2, 50 ng μL^–1 of IS-CXEGFP was injected with or without MegaN at GV or after IVF (EGFP+GV, EGFP–GV, EGFP+IVF, EGFP–IVF). Finally, IS-CXEGFP digested or not by MegaN (EGFP+ or EGFP–) and incubated with cumulus cells for 5 min were intracytoplasmatically injected into enucleated oocytes. Embryos were cultured in SOF. Rates of blastocysts and egfp expression were evaluated. Data was analysed by Fisher's test (P < 0.05). In Experiment 1, no differences in blastocyst rates were observed between the 3 IS-PAXGFP concentrations; however, except for 25+ and 50+, blastocysts rates were lower than after IVF [13/69 (18%), 22/98 (22%), 25/89 (28%), 19/91 (20%), 22/80 (27%), 16/71 (22%) and 31/80 (38%) for 15+, 15–, 25+, 25–, 50+, 50– and IVF, respectively]. Egfp expression was higher for 50– than for other groups, except for 25–, which did not statistically differ [9/69 (13%), 12/98 (12%), 19/89 (21%), 24/91 (26%), 16/80 (20%) and 27/71 (38) for 15+, 15–, 25+, 25–, 50+ and 50–, respectively). We observed higher cleavage and blastocyst rates after injection post IVF as compared with GV (P < 0.05). Egfp expression rates were also higher after injection post IVF, but no differences were found between digestion or not with MegaN [19/95 (20%), 23/95 (24%), 79/115 (68%) and 69/119 (57%) for EGFP+GV, EGFP–GV, EGFP+IVF and EGFP–IVF, respectively]. Egfp blastocyst over total blastocyst rates did not differ between injection at GV or post IVF [3/4 (75%), 2/3 (66%), 32/40 (80%) and 35/46 (76%) for EGFP+GV, EGFP–GV, EGFP+IVF and EGFP–IVF, respectively]. After cloning, no differences were found between EGFP+ and EGFP– in blastocysts [10/57 (17%) and 13/70 (18%) for EGFP+ or EGFP–], or in Day 4 egfp expression (23/57 (40%) and 24/70 (34%), respectively]. Expression of blastocysts after cloning was high [9/10 (90%) and 10/13 (76%) for EGFP+ and EGFP–, respectively]. In conclusion, MegaN transgenesis can produce bovine transgene-expressing embryos, although no differences were observed between digestion or not with MegaN. Injection at GV resulted in transgene-expressing embryos. Cloning with donor cells briefly incubated with MegaN resulted in the highest transgenesis rates. MegaN transgenesis could be useful for transgenic live mammal production, as observed in Xenopus.