Archives of Medical Research 32 (2001) 609–613 0188-4409/01 $–see front matter. Copyright © 2001 IMSS. Published by Elsevier Science Inc. PII S0188-4409(01)00324-1 REVIEW ARTICLE Nuclear Transfer Technology in Mammalian Cloning Don P. Wolf, a,b Shoukhrat Mitalipov a and Robert B. Norgren, Jr. c a Division of Reproductive Sciences, Oregon Regional Primate Research Center, Beaverton, OR, USA b Departments of Obstetrics/Gynecology and Physiology/Pharmacology, Oregon Health Sciences University (OHSU), Portland, OR, USA c Department of Cell Biology and Anatomy, University of Nebraska, Omaha, NE, USA Received for publication June 5, 2001; accepted June 6, 2001 (01/084). The past several years have witnessed remarkable progress in mammalian cloning using nuclear transfer (NT). Until 1997 and the announcement of the successful cloning of sheep from adult mammary gland or fetal fibroblast cells (1), our working assumption was that cloning by NT could only be accomplished with relatively undifferentiated embryonic cells. Indeed, live offspring were first produced by NT over 15 years ago from totipotent, embryonic blastomeres derived from early cleavage-stage embryos. However, once begun, the progression to somatic cell cloning or NT employing differentiated cells as the source of donor nuclei was meteoric, initially involving differentiated embryonic cell cultures in sheep in 1996 (2) and quickly thereafter, fetal or adult somatic cells in sheep, cow, mouse, goat, and pig. Several recent reviews provide a background for and discussion of these successes (3–6). Here we will focus on the potential uses of reproductive cloning along with recent activities in the field and a discussion concerning current interests in human re- productive and therapeutic cloning. © 2001 IMSS. Published by Elsevier Science Inc. Key Words: Reproductive cloning, Therapeutic cloning, Nuclear transfer, Mammals. The Technology Nuclear transfer in its current guise first involves the pro- duction of an enucleated egg, called a cytoplast, whose re- sponsibility it is to reprogram the donor nucleus. Metaphase II oocytes usually used for this purpose can be obtained from either in vitro or in vivo sources. The former are most convenient and prevalent with domestic species in which in vitro maturation of oocytes recovered from slaughterhouse ovaries can lead to the recovery of hundreds of viable metaphase II oocytes. In most other species, in vivo matura- tion is best, requiring that egg donors be subjected to ova- rian stimulation and surgical oocyte retrieval. Enucleation or the removal of the metaphase spindle with its associated maternal chromosomes from the mature unfertilized oocyte involves micromanipulation. The zona pellucida is pene- trated and the first polar body, used as a marker for the metaphase spindle, is aspirated into a micropipette along with approximately 10% of the cytoplasm immediately be- low the first polar body. A continuous membrane surrounds the polar body and the aspirated cytoplasm in such a way that when the pipette is slowly withdrawn, the egg plasma membrane is stretched until it breaks, much like stretching a rubber band beyond the breaking point, leaving an intact cy- toplast. Enucleation, which can be accomplished in high ef- ficiency, is confirmed by the absence of a DNA signal in the cytoplast or by the presence in the enucleating pipette of DNA originating from the polar body and the metaphase plate. A DNA-specific fluorochrome is typically employed with visualization under ultraviolet light. A donor nucleus is then transferred by direct injection of an isolated nucleus or by electrofusion of the cytoplast with an intact donor cell. Although embryonic development can be activated by the electrofusion step in some species, it is common to chemi- cally initiate development with the combination of an iono- phore and a protein kinase inhibitor. The resultant NT em- bryo is placed in culture for a period of time to assess its viability before eventual transfer into an appropriately syn- chronized recipient. Parameters that can affect the success of NT are, for the most part, empirically defined and include the source of the Address reprint requests to: Don P. Wolf, Ph.D., Division of Reproduc- tive Sciences, Oregon Regional Primate Research Center/OHSU, 505 N.W. 185 th Avenue, Beaverton, OR 97006 USA. Tel.: (+503) 690-5326; FAX: (+503) 690-5384; E-mail: wolfd@ohsu.edu