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Background and Techniques used in Nuclear Transfer Studies at Roslin

1. The techniques involved in producing clones in sheep by nuclear transfer (see fig. 1) require that oocytes are recovered by laparotomy from donor ewes. These ewes are superovulated by the administration of hormone by injection and by insertion of a vaginal tampon. On average, ten oocytes are taken per ewe. The oocyte DNA is removed by microsuction, and DNA from the nuclei of the source cells is then transferred into the oocytes after the cell reproduction has been halted by starving' the cells in a nutrient-poor medium, a process which induces the state of quiescence. The cells are then placed in an electrical field which stimulates cell fusion, and the quiescent nuclear DNA then appears to recommence replication in the new environment. Oocyte donor ewes are used for only one surgical donation and are then returned to the farm.

2. Cells which have so far been used as sources for the DNA to be transferred are:

a) embryos from superovulated ewes, recovered by laparotomy (these may be chromosomally unstable);
b) fetuses taken post-mortem from ewes 26 to 35 days after being naturally mated (usually two fetuses are collected per ewe); and
c) mature cells grown in culture - (such as in the case of Dolly, the sheep, who reportedly derives from a mammary cell line).

3. Following nuclear transfer, the oocytes are cultured in vivo in the ligated oviduct of a live sheep for a period of seven days. One ewe can carry large numbers of oocytes for this purpose. After seven days, the ewe is humanely killed and the developed blastocysts transferred to synchronised recipient sheep, two or three blastocysts to each recipient, by laparotomy. These are then allowed to develop to full term to be delivered.

4. The success rates of these various methods have been variable and generally not high. For example, it is acknowledged that in the series of experiments which resulted in Dolly, twenty-one ewes were confirmed to be pregnant but this resulted in only eight of them producing live lambs. The reasons why the other ewes lost their lambs are not clear but obviously congenital abnormality is a possible cause. Furthermore, in the previous series of experiments which resulted in Megan and Morag, two of the lambs died and, at autopsy, showed signs of developmental abnormalities in the liver and kidney. In contrast, it would appear that cloning might ultimately reduce the number of animals needed to produce a desired transgenic animal since it is possible to screen out those altered cells not expressing the gene of interest to avoid inserting them into recipient ewes. This compares with previous studies using pronuclear injection in which many ewes have been required to become pregnant to produce a single transgenic lamb.