99 EFFICIENCY OF CATIONIC LIPID-BASED DNA TRANSFECTION OF BOVINE IVF-DERIVED ZONA-FREE BLASTOCYSTS

Abstract

We have previously reported that zona pellucida (ZP) removal and cationic lipid-based transfection (lipofection) of 1-cell stage bovine embryos with siRNA or DNA did not affect development to the blastocyst (BL) stage (Bertolini et al. 2006 Reprod. Fertil. Dev. 18, 168–169). Moreover, approximately half of green fluorescent protein (GFP)-transfected embryos showed various levels of fluorescence at the BL stage, which was also recently confirmed by others (O’Meara et al. 2010 Reprod. Fertil. Dev. 22, 224). The objective of this study was to evaluate the effect of the timing of ZP removal on the efficiency of lipofection in Day 8 bovine IVF-derived blastocyst stage embryos. In vitro-derived (IVP) embryos, produced based on (Bertolini et al. 2004 Reproduction 128, 341–354), were allocated to one of three experimental groups: (a) G-1, ZP removal on Day 1 of development, in embryos at the 1-cell stage (7 days before transfection); (b) G-2, ZP removal performed on Day 7 BL (24 h before transfection); and (c) G-3, naturally hatched Day 8 BL. On Day 8 of development, zona-free BL from G1, G2, and G3 were transfected by the incubation in 5 μL Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and 12 μg DNA (pEGFP, Clontech Laboratories, Mountain View, CA, USA) in 100 μL Opti-MEM I (Invitrogen), at 39°C for 30 to 60 min. Transfected embryos were cultured for 24 h, when qualitative and quantitative assessments of the transfection efficiency were done by fluorescence microscopy. Positively-transfected BL were DNA-stained for the estimation of the total GFP-positive cells and total cell numbers (ANOVA and Tukey’s test, P < 0.05), and for the estimation of the proportion of GFP-positive cells in each embryonic cell lineage (chi-square test; P < 0.05). Embryos showing at least one GFP-positive cell were qualitatively considered transfected. Cleavage rate on Day 2 (144/197, 73.1%) and BL rate on Day 7 (122/197, 61.9%) were similar between groups (chi-square test; P < 0.05). No qualitative differences in transfection were seen between groups (Table 1), with cell transfection being restricted to the embryonic trophectodermal (TE) cell lineage. Embryo developmental kinetics was slightly delayed in G-1, which also had lower proportion of GFP-positive cells per total TE cell number (GFP/TE) when compared with results with more developed G-2 and G-3 BL. In fact, the number and proportion of GFP-positive cells per embryo appeared to increase more in a stage-dependent manner, rather than due to the time intervals used for ZP removal (Day 1 or Day 7). In summary, lipofection was effective for the transfection of bovine BL, but at a rather low efficiency on an embryo basis. Such procedure may be useful in studies requiring the delivery of nucleic acids specific to the trophectodermal embryonic cell lineage. Table 1.Efficiency of cationic lipid-based DNA transfection of bovine IVF-derived zona-free blastocysts Supported by CNPq and FINEP/Brazil.