Development of a highly efficient method for the production of cloned animals in the agricultural and biomedical fields

The broad use of cloning has been hindered by its relative high cost due to low developmental rates and high rates of abnormalities for embryos/fetuses. In the biomedical field cloning could greatly reduce the costs associated with the production of transgenic livestock and offers the only current approach for gene targeting in livestock. Additionally, in the agricultural field the utilization of cloning would allow for increased genetic selection, recovery of lost genetics, and opportunities for genetic modification of production traits. Improvements to the current protocols utilized for cloning would allow for a broader usage of the technology.; The purpose of this study was to establish an efficient protocol for the production of cloned swine. Additionally phenotypic variation among cloned and age, sex and breed matched controls were compared to determine likely variation due to epigenetics. The protocol was then tested using transgenic cell lines produced by different approaches and on cell lines from adult animals to determine the effect of these variables on the ability to clone animals. The final experiment was conducted to determine if the low level of success encountered when utilizing adult cells could be counteracted by pretreatment of donor cells with chemicals capable of modifying chromatin structure.; The protocol developed for the cloning of swine was much more efficient than any protocols previously reported utilizing fetal cells. Analysis of phenotypic differences in pigs produced by cloning compared with natural breeding showed little statistical difference in variance among cloned and control animals. Interestingly, some phenotypic differences where noted in the clones, including hair appearance, teat numbers and skin morphology, possibly the result of epigenetic abnormalities from the cloning process. Further testing of the protocol utilizing various methods for production of transgenic cell lines and animals from adult cells demonstrated the applicability of the protocol across fields. Testing of the chemical treatments 5 aza-C and TSA on donor cells to modify chromatin structure showed a decrease in cloning efficiencies in both cattle and pigs. In contrast the combination of 5aza-C/TSA had no effect on pregnancy rates compared to the control.