title: Production of chimeric embryos by aggregation of bovine egfp eight-cell stage blastomeres with two-cell fused and asynchronic embryos.
abstract: Embryo disaggregation allows the production of two to four identical offspring from a single cow embryo. In addition, embryo complementation has become the technique of choice to demonstrate the totipotency of embryonic stem cells and induced pluripotent stem cells. Therefore, the aim of this study was to generate a new and simple method by aggregation in the well-of-the-well system to direct each single enhanced green fluorescent protein (egfp) eight-cell blastomere derived from bovine in vitro fertilization embryos to the inner cell mass (ICM) of chimeras produced with fused and asynchronic embryos. To this end, the best conditions to generate in vitro fertilization-fused embryos were determined. Then, the fused (F) and nonfused (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 two pulses of 40 V. The 2ASF and 2ASNF groups showed the best number of blastocysts expressing the EGFP protein (48% and 41%, respectively). Furthermore, 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 embryonic stem cells and induced pluripotent stem cells.
authors.affiliation: Laboratorio de Biotecnología Animal, University of Buenos Aires, Buenos Aires, Argentina.
authors.forename: M I
authors.initials: MI
authors.lastname: Hiriart
authors.forename: R J
authors.initials: RJ
authors.lastname: Bevacqua
authors.forename: N G
authors.initials: NG
authors.lastname: Canel
authors.forename: R
authors.initials: R
authors.lastname: Fernández-Martín
authors.forename: D F
authors.initials: DF
authors.lastname: Salamone
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001757:Blastomeres / Q000166:cytology* / Q000378:metabolism*; D002417:Cattle / Q000196:embryology / Q000235:genetics / Q000378:metabolism; D002459:Cell Fusion / Q000662:veterinary; D002478:Cells, Cultured; D002678:Chimera / Q000196:embryology*; D002970:Cleavage Stage, Ovum / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D019976:Cloning, Organism / Q000379:methods / Q000662:veterinary*; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian; D047108:Embryonic Development / Q000235:genetics; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D008297:Male
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Aggregation; Bovine; Chimera; Transgene