A unique method to produce transgenic embryos in ovine, porcine, feline, bovine and equine species.
Abstract: Transgenesis is an essential tool in many biotechnological applications. Intracytoplasmic sperm injection (ICSI)-mediated gene transfer is a powerful technique to obtain transgenic pups; however, most domestic animal embryos do not develop properly after ICSI. An additional step in the protocol, namely assistance by haploid chemical activation, permits the use of ICSI-mediated gene transfer to generate transgenic preimplantation embryos in a wide range of domestic species, including ovine, porcine, feline, equine and bovine. In the present study, spermatozoa from five species were coincubated with pCX-EGFP plasmid and injected into metaphase II oocytes. The chemical activation protocol consisted of ionomycin plus 6-dimethylaminopurine. We detected high proportions of fluorescent EGFP embryos for all five species (23-60%), but with a high frequency of mosaic expression (range 60-85%). To our knowledge, this is the first study to produce exogenous DNA expression in feline and equine embryos. Chemical activation reduces the lag phase of egfp expression in ovine embryos. Our results show that this unique method could be used to obtain ovine, porcine, feline, bovine and equine transgenic preimplantation embryos.
Publication Date: 2008-10-10 PubMed ID: 18842176DOI: 10.1071/rd07172Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research article introduces a unique method of generating transgenic preimplantation embryos in several domestic species like ovine, porcine, feline, equine, and bovine via Intracytoplasmic sperm injection (ICSI)-mediated gene transfer and haploid chemical activation.
Background
- The study focuses on transgenesis, a crucial tool used in multiple biotechnological applications.
- While ICSI-mediated gene transfer is an effective technique for producing transgenic offspring, most domestic animal embryos do not develop successfully post this procedure.
- As a coping strategy, the researchers introduced an additional step in the process, assistance via haploid chemical activation, which allows the usage of ICSI-mediated gene transfer to create transgenic preimplantation embryos in several domestic species.
Methodology
- The researchers incubated spermatozoa from five different species with the pCX-EGFP plasmid and injected this into metaphase II oocytes.
- The chemical activation protocol comprised ionomycin plus 6-dimethylaminopurine.
Results
- The outcome showed a high proportion of embryos fluorescing EGFP for all the five species, ranging between 23% and 60%. However, there was a high frequency of mosaic expression between 60% and 85%.
- Feline and equine embryos exhibited exogenous DNA expression for the first time, to the extent of the researchers’ knowledge.
- Chemical activation in squashing ovine embryos cut down the egfp expression delay phase.
Conclusion
- Based on the data from the experiment, the researchers concluded that this novel method could effectively generate ovine, porcine, feline, bovine, and equine transgenic preimplantation embryos.
Cite This Article
APA
Pereyra-Bonnet F, Fernández-Martín R, Olivera R, Jarazo J, Vichera G, Gibbons A, Salamone D.
(2008).
A unique method to produce transgenic embryos in ovine, porcine, feline, bovine and equine species.
Reprod Fertil Dev, 20(7), 741-749.
https://doi.org/10.1071/rd07172 Publication
Researcher Affiliations
- Facultad de Agronoma, Universidad de Buenos Aires, Av. San Martn 4453, C1417 Buenos Aires, Argentina.
MeSH Terms
- Animals
- Animals, Genetically Modified / embryology
- Animals, Genetically Modified / genetics
- Cats
- Cattle
- Cloning, Organism / methods
- Cloning, Organism / veterinary
- Embryonic Development
- Female
- Gene Transfer Techniques / veterinary
- Green Fluorescent Proteins / genetics
- Horses
- Male
- Pregnancy
- Recombinant Proteins / genetics
- Sheep
- Species Specificity
- Sperm Injections, Intracytoplasmic / methods
- Sperm Injections, Intracytoplasmic / veterinary
- Swine
Citations
This article has been cited 15 times.- Popova J, Bets V, Kozhevnikova E. Perspectives in Genome-Editing Techniques for Livestock. Animals (Basel) 2023 Aug 10;13(16).
- Hisey EA, Ross PJ, Meyers S. Genetic Manipulation of the Equine Oocyte and Embryo. J Equine Vet Sci 2021 Apr;99:103394.
- Ratner LD, La Motta GE, Briski O, Salamone DF, Fernandez-Martin R. Practical Approaches for Knock-Out Gene Editing in Pigs. Front Genet 2020;11:617850.
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