Porcine transgenesis by intracytoplasmic injection of oolema vesicles co incubated with transgene

LUCHETTI C; BEVACQA R; WILLIS M ; FERRARIS S; VITULLO A; SALAMONE D.

Vancouver

Congreso; International Congress on Animal Reproduction (ICAR); 2012

International Congress on Animal Reproduction (ICAR)

Porcine transgenesis is an essential tool in agriculture, pharmacology and medicine. There is a wide variety of methods in pig transgenesis but they show some limitations. The aim of this work was to evaluate the use of oolema vesicles in the intracytoplasmic injection of a transgene for obtaining GFP+ porcine embryos, with the purpose of developing a simple and economic method in pig transgenesis. Recently, we described an efficient transgenesis system in bovines using co incubation with oolema vesicles with the transgene, and its subsequent intracytoplasmic injection. To test the use of vesicles technique in porcine activated oocytes we compared intracytoplasmic injection with (i) the plasmid alone or (ii) the plasmid co incubated with vesicles. The plasmid used is pCX-EGFP, linearized with HINdIII enzyme, at 30 ng/ll. Pig oocytes matured in vitro were activated by an electric pulse followed by treatement of 6-DMAP. Embryos were cultured in SOF medium at 39C, 5% CO2. Experimental groups were: control (without injection; N = 109) – pCXEGFP naked (injected only with pVp + plasmid; N = 58) – pCXEGFP vesicles (injected with pVp + vesicles + plasmid; N = 76) – SHAM pVp (injected only with pVp; N = 16) – SHAM vesicles (injected with pVp + vesicles; N = 20). Rates of blastocysts at day 7 and eGFP expression under blue light were evaluated. Data were analyzed by Fisher’s test (p < 0.05). Both groups injected with plasmid presented a lower % of cleavage respect to control [control 94%; pCX-EGFP naked 84% (p < 0.05) and pCX-EGFP vesicles 80% (p < 0.005)]; no differences in % of cleavage were observed between both SHAM groups and the other experimental groups. No differences were observed in blastocyst rates between the different groups. eGFP expression was higher in pCX-EGFP vesicles than in pCX-EGFP naked [pCX-EGFP vesicles 74% vs. pCX-EGFP naked43% (p < 0.005)]. No differences were observed in the % of blastocysts expressing the transgene [pCX-EGFP vesicles 100%; pCX-EGFP naked 33% (p > 0.05)]. These results suggest that intracytoplasmic injection of pCX-EGFP linearized with HINdIII enzyme, both naked plasmid and co incubated with vesicles, affects early development of porcine parthenogenetic embryos; that is reflected in the lower % of cleavage of pCX-EGFP groups compared to control. Respect to eGFP expression, evaluation of genomic integration remains to be tested but these results suggest that the use of vesicles in the intracytoplasmic injection is a useful tool for obtaining GFP+ porcine embryos. Key Words: Pig, transgenesis, parthenogenetic, vesicles