Reprogramming of Ovine Somatic Cells with Xenopus laevis Oocyte Extract Prior to SCNT Improves Live Birth Rate

Abstract

Abstract

The birth of live animals following somatic cell nuclear transfer (SCNT) has demonstrated that oocytes can reprogram the genome of differentiated cells. However, in all species the frequency of development of healthy offspring is low; for example, in sheep, approximately only 5% of blastocysts transferred develop to term, and less than 3% develop to adulthood. Such low efficiencies, coupled with the occurrence of developmental abnormalities, have been attributed to incomplete or incorrect reprogramming. Cytoplasmic extracts from both mammalian and amphibian oocytes can alter the epigenetic state of mammalian somatic nuclei and reprogram gene expression to more resemble that of pluripotent cells. Therefore, it may be possible to increase the frequency or success of normal development by pretreating somatic cells to be used as nuclear donors prior to SCNT. In the present study, permeabilized ovine fetal fibroblasts were pretreated with a cytoplasmic extract produced from germinal vesicle (GV) stage Xenopus laevis oocytes. No increase in the frequency of development to blastocyst stage or pregnancy rate was observed; however, live birth and survival rates were significantly improved. Development to term of blastocysts transferred increased from 3.1% in the control group, to 14.7% in the treated group (a 4.7-fold increase), and even though the subsequent survival of lambs produced from treated cells was reduced by 60%, the percentage of lambs surviving to adulthood of blastocysts transferred (5.9%) increased 1.9-fold compared to controls. This study is the first to report the birth of live offspring and an increase in cloning efficiency, after crossspecies pre-reprogramming using Xenopus GV stage oocyte extract.