Production of cloned pigs from in vitro systems

Abstract

Here we describe a procedure for cloning pigs by the use of in vitro culture systems. Four healthy male piglets from two litters were born following nuclear transfer of cultured somatic cells and subsequent embryo transfer. The initiation of five additional pregnancies demonstrates the reproducibility of this procedure. Its important features include extended in vitro culture of fetal cells preceding nuclear transfer, as well as in vitro maturation and activation of oocytes and in vitro embryo culture. The cell culture and nuclear transfer techniques described here should allow the use of genetic modification procedures to produce tissues and organs from cloned pigs with reduced immunogenicity for use in xenotransplantation.

Effects of Donor Fibroblast Cell Type and Transferred Cloned Embryo Number on the Efficiency of Pig Cloning

“…Success in production of cloned pigs from somatic cells was first reported almost at the same time in 2000 by three independent groups (Betthauser et al, 2000;Onishi et al, 2000;Polejaeva et al, 2000). Since then, the somatic cell nuclear transfer (SCNT) technique has been used to amplify superior pigs or has been applied in combination with genetic modifications to generate different types of transgenic or knockout pigs (Klymiuk et al, 2010;Prather et al, 2008;Schmidt et al, 2010;Vajta and Callesen 2012).…”

Pig transgenesis by Sleeping Beauty DNA transposition

“…With the successful cloning of animals by somatic cell nuclear transfer (SCNT), it is now possible to produce transgenic pigs from genetically engineered somatic donor cells. In SCNT, reported for a variety of animal species including the mouse (Wakayama et al 2000; Yanagimachi 1999), the sheep (Wilmut et al 1997), the cow (Cibelli et al 1998;Kato et al 1998;Kubota et al 2000), and the pig (Betthauser et al 2000;Onishi et al 2000;Polejaeva et al 2000), the nucleus from a single differentiated somatic cell is transferred into an enucleated oocyte (unfertilized egg cell), and the reconstructed embryo is subsequently transferred to a surrogate mother. This procedure allows modification of the somatic donor cell in culture by transgene insertion or introduction of loss-offunction gene knockout mutations by homologous recombination.…”

Expression of X‐linked genes in deceased neonates and surviving cloned female piglets

“…Since the creation of Dolly, the first mammal cloned from a differentiated adult somatic cell, production of live offspring through nuclear transfer (NT) of somatic cells has been successful in a variety of species, ranging from small laboratory mice to large domestic animals (Betthauser et al 2000;Cibelli et al 1998;Galli et al 2003;Kato et al 1998;Kubota et al 2000;Onishi et al 2000;Polejaeva et al 2000;Senda et al 2004;Shin et al 2002;Wilmut et al 1997). Somatic cell nuclear transfer revealed the extraordinary ability of the oocyte to reprogram the differentiated somatic genome to a totipotent state (Eggan et al 2004;Humpherys et al 2002).…”