Production of calves from G1 fibroblasts

Abstract

Since the landmark study of Wilmut et al. (Wilmut et al. in Nature 385:810, 1997) describing the birth of a cloned lamb derived from a somatic cell nucleus, there has been debate about the donor nucleus cell cycle stage required for somatic cell nuclear transfer (NT). Wilmut et al. (Wilmut et al. in Nature 385:810, 1997) suggested that induction of quiescence by serum starvation was critical in allowing donor somatic cells to support development of cloned embryos. In a subsequent report, Cibelli et al. (Cibelli in Science 280:1256, 1998) proposed that G0 was unnecessary and that calves could be produced from actively dividing fibroblasts. Neither study conclusively documented the importance of donor cell cycle stage for development to term. Other laboratories have had success with NT in several species (Wilmut et al. in Nature 385:810, 1997, Cibelli in Science 280:1256, 1998, Baguisi in Nat. Biotechnol. 17:456, 1999, Kubota in Proc. Natl. Acad. Sci. USA 973:990, 2000, Wakayama et al. in Nature 23:369, 1998, Wells et al. in Biol. Reprod. 60:996, 1999, Polejaeva in Nature 407:505, 2000) and most have used a serum starvation treatment. Here we evaluate methods for producing G0 and G1 cell populations and compare development following NT. High confluence was more effective than serum starvation for arresting cells in G0. Pure G1 cell populations could be obtained using a “shake-off” procedure. No differences in in vitro development were observed between cells derived from the high-confluence treatment and from the “shake-off” treatment. However, when embryos from each treatment were transferred to 50 recipients, five calves were obtained from embryos derived from “shake-off” cells, whereas no embryos from confluent cells survived beyond 180 days of gestation. These results indicate that donor cell cycle stage is important for NT, particularly during late fetal development and that actively dividing G1 cells support higher development rates than cells in G0.