High rates of bovine blastocyst development after ICSI-mediated gene transfer assisted by chemical activation.
In order to establish conditions for intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) in cattle, various aspects of fertilization and embryonic development were assessed after five activation treatments. Spermatozoa were co-incubated with pCX-EGFP (pCX-enhanced green fluorescent protein gene) plasmid and injected into metaphase II oocytes, which were then treated with ionomycin (Io), before further activation with the following agents: 6-dimethylaminopurine (Io-DMAP), additional Io plus DMAP (2Io-DMAP), Io alone (2Io), ethanol (Io-EtOH), or strontium chloride (Io-SrCl2). Fertilization rates at 16 h after ICSI, presence of a condensed spermatozoon head on Day 4 (Day 0 = ICSI), blastocyst and EGFP expression rates on Day 7, and Oct-4 pattern of Day 8 blastocysts were evaluated. Fertilization rates did not differ significantly among treatments. All (100%) of EGFP-positive embryos resulted from ICSI fertilization, whereas at least 60% of EGFP-negative embryos (>4 cells) had a condensed sperm head. Blastocyst rates after 2Io-DMAP were not significantly different from Io-DMAP or Io-EtOH, but they were higher than 2Io or Io-SrCl2 treatments (25.9, 18.7, 14.7, 9.4, and 10.9% respectively; P < 0.05). Transgene expression rates were higher for Io-DMAP, 2Io-DMAP and Io-SrCl2 than for 2Io and Io-EtOH (52.3, 53.0, 42.8, 28.2, and 29.4% respectively; P < 0.05). Over 80% of the blastocysts expressed egfp protein. In conclusion, ICSI-MGT was a powerful technique to produce bovine embryos that expressed the EGFP transgene. Moreover, the actual efficiency of ICSI-MGT could be readily evaluated by this method, which uses a marker expressed early in embryo development.
Bevacqua
Romina J
RJ
Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Av. San Martín 4453, C1417 Buenos Aires, Argentina.
Pereyra-Bonnet
Federico
F
Fernandez-Martin
Rafael
R
Salamone
Daniel F
DF
D000818:Animals; D030801:Animals, Genetically Modified; D001755:Blastocyst / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D002417:Cattle; D002452:Cell Count; D002478:Cells, Cultured; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian / Q000166:cytology; D047108:Embryonic Development / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D005260:Female; D018014:Gene Transfer Techniques / Q000662:veterinary*; D049452:Green Fluorescent Proteins / Q000235:genetics; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008297:Male; D020554:Sperm Injections, Intracytoplasmic / Q000662:veterinary*; D013268:Stimulation, Chemical
D007476:Ionophores; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D015759:Ionomycin