Nuclear transfer (NT) is an inefficient method of replicating animals. Originally, cleavage-stage embryos were used, usually before compaction of the blastomeres, and this meant that it was only possible to produce small numbers of identical animals (1). This changed in 1995, when animals were produced from cells that had been grown in culture (2), greatly simplifying the production of identical animals. These experiments were conducted on sheep oocytes

From: Methods in Molecular Biology, vol. 325: Nuclear Reprogramming: Methods and Protocols Edited by: S. Pells © Humana Press Inc., Totowa, NJ

because they were relatively cheap to use and because efficient methods of handling the animals existed at the Roslin Institute. The cells used as nuclear donors were cultured from the inner cell mass (ICM) of sheep embryos. The following year, similar experiments were performed using three different cell types: ICM cells, fetal cells, and cells from the udder of a 6-yr-old adult animal. The three cell lines produced four lambs, two lambs, and one lamb, respectively; the animal produced from the udder cells was "Dolly" (3).

Current methods of NT have not changed to any great extent, but the process has become easier as a result of the development of specific sheep culture medium (4) and the production of specialized tools and equipment. NT is still inefficient with somatic cell nuclear transfer (SCNT) efficiency running at 15%, but it is much easier because of the development of new technology (5,6).

NT consists of the removal of the maternal chromosomes and the replacement of these chromosomes with those from a diploid cell. The oocytes are usually used at the metaphase II stage; they have their maternal deoxyribo-nucleic acid (DNA) removed using cytoskeletal inhibitors and a DNA-specific dye. A cell is placed in the perivitelline space and the cell is then fused to the cytoplast using an electric current. The reconstructed embryo is then activated and allowed to develop before the transfer into a surrogate mother (7).

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