Simple gene transfer technique based on I-SceI meganuclease and cytoplasmic injection in IVF bovine embryos.

Although transgenic methods in mammals are inefficient, an easy and highly efficient transgenesis system using I-SceI meganuclease (intron-encoded endonuclease from S. cerevisiae) was recently described in Xenopus. The method consisted of injection into fertilized eggs of an I-SceI reaction mixture with a plasmid DNA carrying the transgene, flanked by the meganuclease recognition sites (pIS). In the present study, the effects of I-SceI on gene transfer were tested apparently for the first time in mammals, in particular, in cattle. Various conditions were evaluated, including three concentrations of the plasmid pIS Pax6egfp, carrying I-SceI recognition sites flanking egfp under Pax6 promoter and two injection times (before IVM and after IVF) of pIS CAGegfp, carrying I-SceI sites fanking egfp under CAG promoter. In addition, the quantity of transgene was measured using quantitative polymerase chain reaction, and presence of transgene signals was evaluated using fluorescence in situ hybridization analysis. Transgene expression rates were higher (P < 0.05) for groups treated after IVF (79.1%, 91/115 and 63.0%, 75/119) than before IVM (32.6%, 31/95 and 34.7%, 33/95), with and without I-SceI, respectively. Interestingly, injection with pIS plus I-SceI after IVF increased frequency (P < 0.05) of nonmosaic transgene-expressing embryos (58.3%, 42/72 vs. 29.7%, 25/84) for pIS plus I-SceI and pIS alone. Based on fluorescence in situ hybridization analysis, injection with I-SceI increased (P < 0.05) the proportion of embryos with transgene signals in all blastomeres compared with pIS alone (44.0%, 11/25 vs. 6.9%, 2/29) for pIS plus I-SceI and pIS alone. In addition, transgene copy number was numerically higher for the group treated with pIS plus I-SceI compared with pIS alone. In conclusion, I-SceI gene transfer increased transgene signals in bovine embryos.

Production of IVF transgene-expressing bovine embryos using a novel strategy based on cell cycle inhibitors.

The objective was to evaluate the effects of cell cycle inhibitors (6-dimethylaminopurine [DMAP], and dehydroleukodine [DhL]) on transgene expression efficiency and on mosaic expression patterns of IVF bovine zygotes cytoplasmically injected with oolema vesicles coincubated with transgene. The DNA damage induced by the transgene or cell cycle inhibitors was measured by detection of phosphorylated histone H2AX foci presence (marker of DNA double-stranded breaks). Cloning of egfp blastomeres was included to determine continuity of expression after additional rounds of cellular division. The pCX-EGFP [enhanced green fluorescent protein gene (EGFP) under the chimeric cytomegalovirus IE-chicken-ß-actin enhancer promoter control] gene plasmid (50 ng/µL) was injected alone (linear or circular exogenous DNA, leDNA and ceDNA, respectively) or associated with ooplasmic vesicles (leDNA-v or ceDNA-v). The effects of 2 mm DMAP or 1 µm DhL for 6 h (from 15 to 21 h post IVF) was evaluated for groups injected with vesicles. The DMAP increased (P < 0.05) egfp homogenous expression relative to transgene alone (21%, 18%, and 11% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively) and also increased (P < 0.05) the phosphorylated histone H2AX foci area. Expression of egfp was higher (P < 0.05) for linear than for circular pCX-EGFP, and egfp blastocyst rates were higher (P < 0.05) for groups injected with linear transgene coincubated with vesicles than for linear transgene alone (95%, 77%, 84%, and 52% for leDNA-v + DMAP, leDNA-v + DhL, leDNA-v, and leDNA, respectively). Moreover, DMAP tended to improve egfp blastocysts rates for both circular and linear transgenes. Based on fluorescent in situ hybridization (FISH) analysis, there was evidence of integration in egfp embryos. Finally, clones derived from leDNA-v + DMAP had the highest egfp expression rates (96%, 65%, and 65% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively). Transgenesis by cytoplasmic injection of leDNA-v + DMAP is a promising alternative for transgenic animal production.