2 NEW IVF TRANSGENESIS STRATEGY IN BOVINE USING CELL CYCLE INHIBITORS AND MOSAICISM REVERSION BY CLONING

All the techniques available for transgenic animal production are inefficient. A new transgenic embryo production strategy was developed, consisting on injection of oolema vesicles coincubated with DNA into fertilized embryos. To improve this technique, we evaluated the effects of 1) linear and circular covalently closed plasmid structures; 2) cell cycle inhibitors (DMAP and Dehydroleucodine, DhL) during first pronuclear phase. The effect of the transgene on DNA double strand breaks (DSBs) was also tested. Transgenic blastomeres produced by these strategies were cloned to reverse mosaicism. To this aim, COCs were IVM and subjected to IVF (Bracket and Oliphant, 1975). Then, presumptive zygotes were injected with oolema vesicles incubated with plasmid (OVIP) or with plasmid alone. The plasmid employed was linearized or circular pCX-EGFP. Two treatments were evaluated: 2 mM DMAP (OVIP+DMAP) or 1 uM DhL (OVIP+DhL) for 6 h (15 to 21 h post IVF). Dynamics of egfp expression were daily evaluated. The DNA DSBs were measured by immunocytochemistry against phosphorylated H2AXγ histone. Cloning of IVF transgenic blastomeres was included for the groups OVIP+DMAP, OVIP and for plasmid alone. Rates of egfp expression were higher for linear than circular pCX-EGFP [58/81(71%), 45/68(66%), 51/84(60%), 34/63(53%) v. 24/72(33%), 30/72(41%), 31/93(33%), 18/53(33%), for OVIP+DMAP, OVIP+DhL, OVIP, and plasmid alone respectively; P < 0.05]. As well, egfp expression was delayed after circular plasmids injection (at day 3 post fertilization, a maximum of 7% for circular and over 40% for linear injected groups). For the groups injected with circular pCX-EGFP, there were not differences in egfp blastocysts rates [16/26(61%); 11/23(47%); 13/28(46%), and 5/11(45%) for OVIP+DMAP, OVIP+DhL, OVIP, and plasmid alone respectively]. Except for the group injected with plasmid alone, linear plasmid structure improved egfp blastocysts rates [21/22(95%); 17/22(77%); 22/26(84%) and 10/19(52%) for OVIP+DMAP, OVIP+DhL, OVIP, and Plasmid alone, respectively]. In all cases, DMAP incubation tended to improve egfp rates. Preliminar confocal images showed higher H2AXγ foci number after OVIP injection previous to DMAP or DhL than for DMAP or DhL alone (mean foci number: 122, 72, and 135 for OVIP+DhL, OVIP+DMAP and OVIP v. 38 and 49 for IVF+DhL or +DMAP). Homogenous egfp expression was detected in up to 22% of embryos used as donors for cloning. All cloned blastocysts derived from OVIP+DMAP or OVIP were transgenic (total n = 10). For plasmid alone injection, 20% (1/5) blastocysts lost egfp expression after cloning. Homogenous expression was detected in 100% of OVIP and OVIP+DMAP cloned embryos (33/33 and 40/40), statistically different to plasmid alone (32/42; 76%). In conclusion, the best conditions for IVF mediated transgenesis were OVIP injection, using linearized plasmid structures, followed by DMAP. The presence of the transgene was related to an increase in DNA DSBs. Cloning of egfp blastomeres resulted in mosaicism reversion. This new IVF mediated transgenesis technique represents an interesting alternative for transgenic animal production.