Production of calves from G1 fibroblasts

Since the landmark study of Wilmut et al.(1) describing the birth of a clon ed lamb derived from a somatic cell nucleus, there has been debate about th e donor nucleus cell cycle stage required for somatic cell nuclear transfer (NT). Wilmut et al.(1) suggested that induction of quiescence by serum sta rvation was critical in allowing donor somatic cells to support development of cloned embryos. In a subsequent report, Cibelli et al.(2) proposed that G0 was unnecessary and that calves could be produced from actively dividin g fibroblasts. Neither study conclusively documented the importance of dono r cell cycle stage for development to term. Other laboratories have had suc cess with NT in several species(1-7), and most have used a serum starvation treatment. Here we evaluate methods for producing G0 and G1 cell populatio ns 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 t reatment and from the "shake-off" treatment. However, when embryos from eac h treatment were transferred to 50 recipients, five calves were obtained fr om embryos derived from "shake-off" cells, whereas no embryos from confluen t 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.