430 INJECTION OF CELLS OR THEIR PARTS AFTER A SHORT EXPOSURE TO PLASMID CONSTRUCTS INDUCES TRANSGENESIS IN OVINE AND BOVINE EMBRYOS

Abstract

As animal transgenesis is an essential tool in medicine and agriculture, it is necessary to understand the mechanisms in order to develop novel methods of transgenesis. We intended to determine if the injection of cells or their parts into metaphase II (MII) oocytes after incubating with exogenous DNA can induce transgenesis in embryos. Sperm cells for intracytoplasmic sperm injection (ICSI) in ovine and cumulus cells for NT in bovine were incubated with pCX-EGFP plasmid (5 to 50 ng μL^-1) for 5 min in 2.8% Na citrate at 0°C before transfer into a 10% polyvinylpyrrolidone (PVP) droplet and injection into MII oocytes (previously enucleated in NT). In both species, oolemma-ooplasmic vesicles (OOV) of 9 μm diameter obtained from MII oocytes by microsurgery were directly incubated in PVP droplet with same pCX-EGFP concentration. As a control group, pCX-EGFP suspension from PVP droplet was injected into MII oocytes. The NT bovine zygotes were activated in 5 μM ionomycin (Io) for 4 min followed by 1.9 mM DMAP immediately for 3 h. In ICSI ovine, the treatment with DMAP was applied 3 h later. Injected oocytes of OOV and controls were activated as NT in bovine and as ICSI in ovine. Expression of EGFP was determined with fluorescence microscopy under blue light (488 nm) at Days 4 to 7, and data were analyzed by Fisher test (P = 0.05). A group of NT, ICSI, OOV, and control presumptive zygotes were treated with FITC-labeled bovine fragments (100-300 bb) DNA in order to determine the binding sites with exogenous DNA by laser confocal microscope analysis. Quantitative PCR (qPCR) was performed to determine pCX-EGFP copy number at 0, 8, 16, and 24 h after Io in all ovine treatments. Embryos expressing EGDP from all techniques were subjected to FISH with rhodamine-labeled pCX-EGFP plasmid as a probe. In ovine, ICSI and OOV injection green embryos at Day 4 [58% (61/105) v. 21.5% (8/38); P < 0.05] and green blastocysts at Day 7 [71.8% (23/32) v. 66.6% (2/3)] were obtained, respectively. In bovine, green embryos [49.2% (34/69) v. 29.7% (14/47); P < 0.05] and green blastocysts [95.8% (23/24) v. 25.0% (2/8); P < 0.05] were produced by NT and OOV injection, respectively. In controls, no green embryos were obtained in ovine (0/47) and only low rates were observed in bovine [3.0% (2/65)]. Confocal images of zygotes showed specific signal only in cumulus cells, spermatozoa, and OOV The qPCR analysis showed similar plasmid copy number/zygote between treatment and times in ovine (range 30 000-300,000). Embryo FISH images showed 1 to 2 specific signals in ICSI and NT interphases of both species and in OOV ovine metaphases, the latter being direct evidence of transgene integration. These results suggest that the injected cells or cellular parts (OOV) dramatically increase transgenesis in ovine and bovine embryos. Until now, the generation of NT and OOV embryos after short exposure to the DNA construction has not been reported. We are performing embryo transfer and at the moment we have a pregnancy derived from ICSI in ewes. In conclusion, the cellular parts/transgene complex may affect exogenous DNA delivery or its interaction with embryo DNA, facilitating the mechanism of transgenesis in mammals.