Abstract
Somatic cell nuclear transfer (SCNT) is a technique by which the nucleus of a differentiated cell is introduced into an oocyte from which its genetic material has been removed by a process called enucleation. In mammals, the reconstructed embryo is artificially induced to initiate embryonic development (activation). The oocyte turns the somatic cell nucleus into an embryonic nucleus. This process is called nuclear reprogramming and involves an important change of cell fate, by which the somatic cell nucleus becomes capable of generating all the cell types required for the formation of a new individual, including extraembryonic tissues. Therefore, after transfer of a cloned embryo to a surrogate mother, an offspring genetically identical to the animal from which the somatic cells where isolated, is born. Cloning by nuclear transfer has potential applications in agriculture and biomedicine, but is limited by low efficiency. Cattle were the second mammalian species to be cloned after Dolly the sheep, and it is probably the most widely used species for SCNT experiments. This is, in part due to the high availability of bovine oocytes and the relatively higher efficiency levels usually obtained in cattle. Given the wide utilization of this species for cloning, several alternatives to this basic protocol can be found in the literature. Here we describe a basic protocol for bovine SCNT currently being used in our laboratory, which is amenable for the use of the nuclear transplantation technique for research or commercial purposes.
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Acknowledgments
We present here the most reliable protocol we know for bovine SCNT. It is the product of cumulative knowledge produced by a vast number of researchers. Some of the techniques here described have likely not been previously published in peer-reviewed journals but were transmitted orally from researcher to researcher. We are indebted to those who came before and helped us design this protocol and we offer an apology to those whose work has not been cited when compiling it. We thank all current and past members of the Cellular Reprogramming Laboratory for contributions toward the establishment of this protocol and to Juan Arechaga, Sebastian Canovas Bernabe and Marcelo Goissis for critical reading of the manuscript. Work in our laboratory was supported by USDA–CSREES competitive grant no 2007-04179 and by the Michigan State University Experiment Station.
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Ross, P.J., Cibelli, J.B. (2010). Bovine Somatic Cell Nuclear Transfer. In: Ding, S. (eds) Cellular Programming and Reprogramming. Methods in Molecular Biology, vol 636. Humana Press. https://doi.org/10.1007/978-1-60761-691-7_10
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DOI: https://doi.org/10.1007/978-1-60761-691-7_10
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