Production of quimeric embryos by aggregation of bovine egfp 8-cell stage blastomeres with two-cell fused and asynchronic embryos

HIRIART MI; BEVACQUA RJ; CANEL N; FERNÁNDEZ Y MARTÍN R; SALAMONE DF

THERIOGENOLOGY

ELSEVIER SCIENCE INC

Lugar: Amsterdam; Año: 2013 vol. 80 p. 357 - 364

0093-691X

Embryo disaggregation allows the production of 2 to 4 identical offspring from a single cow embryo. In addition, embryo complementation has become the technique of choice to demonstrate the totipotency of ES and iPS cells. For those reasons, the aim of this work was to generate a new and simple method to direct each single egfp 8-cell blastomere derived from bovine IVF embryos to the ICM of chimeras produced with fused and asynchronic embryos, by aggregation in the Well of the Well (WOW) system. To this end, the best conditions to generate IVF fused embryos were determined. Then, fused (F) and non-fused (NF) embryos were aggregated in two distinct conditions: synchronically (S), with both transgenic and F embryos produced on the same day; and asynchronically (AS), with transgenic embryos produced one day before F embryos. The highest fusion and blastocysts rates were obtained with 2 pulses of 40V. The 2ASF and 2ASNF groups showed the best number of blastocysts expressing the EGFP protein (48 and 41%, respectively) and moreover, the 2ASF group induced the highest localization rates of the egfp-expressing blastomere in the ICM (6/13, 46% of ICM transgene-expressing blastocysts). This technique will have great application for multiplication of embryos of high genetic value or transgenic embryos and also with the generation of truly bovine ES and iPS cells.