A unique method to produce transgenic embryos in ovine, porcine, feline, bovine and equine species

摘要

Transgenesis is an essential tool in many biotechnological applications. Intracytoplasmic sperm injectionn(ICSI)-mediated gene transfer is a powerful technique to obtain transgenic pups; however, most domestic animal embryosndo not develop properly after ICSI. An additional step in the protocol, namely assistance by haploid chemical activation,npermits the use of ICSI-mediated gene transfer to generate transgenic preimplantation embryos in a wide range ofndomestic species, including ovine, porcine, feline, equine and bovine. In the present study, spermatozoa from five speciesnwere coincubated with pCX-EGFP plasmid and injected into metaphase II oocytes. The chemical activation protocolnconsisted of ionomycin plus 6-dimethylaminopurine.We detected high proportions of fluorescent EGFP embryos for allnfive species (23–60%), but with a high frequency of mosaic expression (range 60–85%). To our knowledge, this is the firstnstudy to produce exogenous DNA expression in feline and equine embryos. Chemical activation reduces the lag phase ofnegfp expression in ovine embryos. Our results show that this unique method could be used to obtain ovine, porcine, feline,nbovine and equine transgenic preimplantation embryos.