Production of Cloned Pigs from Adult Somatic Cells by Chemically Assisted Removal of Maternal Chromosomes1

Abstract

The present study demonstrated that brief treatment of in vitro-matured porcine oocytes with demecolcine results in a membrane protrusion that contains a condensed chromosome mass, which can be easily removed by aspiration. This simple, chemically assisted method for removing maternal chromosomes enabled the production of a large number of nuclear-transferred porcine eggs. The development of eggs whose chromosomes were removed by this procedure following transfer of somatic cell nuclei to the blastocyst stage was not significantly different among groups activated using different procedures (6% to 11%) and was also not different among donor cells of different origins (3% to 9%), except for cumulus cells (0.4%). After transfer of 180 to 341 nuclear-transferred eggs that received somatic cells to 6 recipients, 2 of the recipients produced 8 healthy cloned piglets from the heart cells of a female pig. The chemically assisted method for removing maternal chromosomes was also effective for bovine and rabbit eggs.

Factors Determining the Efficiency of Porcine Somatic Cell Nuclear Transfer: Data Analysis with Over 200,000 Reconstructed Embryos

“…Most previous publications failed to investigate the in vivo development or data collected were insufficient for statistical calculations (Faast et al, 2006;Tomii et al, 2005;Uhm et al, 2000;Yin et al, 2002). Our results confirmed that the capacity of nucleus to be reprogrammed cannot be evaluated exclusively by in vitro development; in contrast, in vivo development may be a more important characteristic.…”

“…Lee et al (2003) reported that more blastocysts were derived from SCNT of FFs than from that of other cells (AFs, cumulus cells, and oviduct cells). Yin et al (2002) reported that, by using FFs, the blastocyst rate was higher than those obtained with adult cells of various origin (cumulus, liver, heart, kidney, muscle, oviduct). The difference may be due mainly to different types of adult-derived donor cells used in our study and those papers.…”

Effect of Postactivation Treatment with Latrunculin A on <i>In Vitro</i> and <i>In Vivo</i> Development of Cloned Embryos Derived from Kidney Fibroblasts of an Aged Clawn Miniature Boar

“…The use of pig kidney fibroblasts as donor cells for SCNT experiment has been reported by several investigators [8,10,37]. However, to our knowledge, we produced the first cloned miniature piglet derived from kidney cells of an aged adult.…”

“…To date, pigs have been produced by SCNT techniques using donor cells derived from fetuses and young pigs [4][5][6][7][8][9][10][11][12][13]. The use of pig kidney fibroblasts as donor cells for SCNT experiment has been reported by several investigators [8,10,37].…”

“…To date, pigs have been cloned with SCNT using donor cells derived from fetal pigs to pigs several years of age. The efficiency of SCNT is influenced by many factors, including quality of recipient oocytes, type of donor cells, reprogramming of donor nuclei and condition of recipient gilts [4][5][6][7][8][9][10][11][12][13]. In particular, the diploid complement retention is one of the most important factors in improving the developmental ability of SCNT embryos [14,15].…”