Nuclear Transfer in the Rhesus Monkey: Practical and Basic Implications1

Abstract

In early 1997, the birth of a lamb after transfer of the nucleus from an adult mammary gland cell into an enucleated oocyte, along with the production of rhesus monkeys by nuclear transfer of embryonic cells, marked a reemergence of the field of mammalian cloning. Clonally derived rhesus monkeys would be invaluable in biomedical research, and the commercial interests in transgenic sheep and cattle propagated by cloning are substantial. Nuclear transfer technology is under consideration in human in vitro fertilization clinics to overcome infertility secondary to advanced maternal age or mitochondrial-based genetic disease. Nuclear transfer involves preparing a cytoplast as a recipient cell, in most cases a mature metaphase II oocyte from which the chromosomes have been removed. A donor nucleus cell is then placed between the zona and the cytoplast, and fusion, as well as cytoplast activation, is initiated by electrical stimulation. Successful reprogramming of the donor cell nucleus by the cytoplast is critical--a step that may be influenced by cell cycle stage. Embryos produced by nuclear transfer are cultured in vitro for several cell divisions before cryopreservation or transfer to the oviduct or uterus of a host mother. The efficiency of producing live young by nuclear transfer in domestic species is low, with a high frequency of developmental abnormalities in both preterm and term animals. However, a number of pregnancies have now been established using fetal cells as the source of donor nuclei. The use of cell lines not only allows large clone sizes but also supports the ability to genetically manipulate cells in vitro before nuclear transfer. Ongoing research focused on the production of clonally derived rhesus monkeys using fetal fibroblasts and embryonic stem cells as the source of donor nuclei will be reviewed.

Clonal Propagation of Primate Offspring by Embryo Splitting

“…identical offspring. Blastomere nuclear transSplits have been frozen and transferred sucfer has been successful [3.8% live fetuses per cessfully; clones with differing birth dates embryos transferred (8)]. Splitting results in have numerous applications: e.g., pheno-31% pregnancies and 8% births, and similar typic analysis of an offspring before propresults were obtained by using four-to eight-agating clones; serial transfer of stem cells cell blastomere nuclear transfer in rhesus, i.e., to address cellular aging beyond life ex-24% (4117) pregnancies and 12% (2117) pectancy; and investigating simultaneous births (8).…”

Long-Acting Depot Formulation of Luprolide Acetate as a Method of Hypothalamic Down Regulation for Controlled Ovarian Hyperstimulation and Oocyte Production in Macaca fascicularis1

“…The long-acting agonist could be administered at any point during the third week of the menstrual cycle, as opposed to Days 21,22, or 23 with the short-acting form. Once menses had occurred, stimulations for this group could be initiated at any point during the month following the single injection.…”

“…Short-acting antagonist down regulation in rhesus monkeys has been reported by Zelinski-Wooten et al [21], with 51% mature oocyte production, as well as by SanchezPartida et al [23], with a 57% maturity rate. In addition, Wolf et al [22] reported an average yield of 16.7 oocytes per retrieval, and Hewitson et al [33] reported an average oocyte yield of 27 oocytes per retrieval and 53.7% maturity using short-acting antagonist down regulation. Cseh et al [28] induced down regulation in baboons with a long-acting agonist similar to Lupron Depot (goserelin acetate) followed by a stimulation protocol.…”

“…This immediate action prevents premature ovulation and lacks the initial stimulatory phenomenon seen with agonist use [20]. This method first appeared in the nonhuman primate literature [21][22][23][24], but during the past two years, it has acquired approval from the U.S. Food and Drug Administration and is now being used clinically [25,26]. This approach decreases the interval of down regulation, because the antagonist is only used for 5-6 days of the cycle, during the latter part of the stimulation period.…”

In Vitro Manipulation of Nonhuman Primate Gametes for Embryo Production and Embryo Transfer.

“…Nuclear transfer was also tried in the rhesus monkey [63,113]. We therefore have begun studies on oocyte activation, pronucleus formation, pronucleus fusion and subsequent embryo development.…”