Live Birth of Somatic Cell-Cloned Rabbits following Trichostatin A Treatment and Cotransfer of Parthenogenetic Embryos

Abstract

Somatic cell nuclear transfer (SCNT) efficiency is still low in rabbit. Previous studies indicated that trichostatin A (TSA) treatment could improve cloning efficiency and term development in the mouse, and cotransfer of parthenogenetic (PA) embryos benefited the pregnancy of cloned embryos in porcine and the mouse. In this study we investigated the effect of TSA treatment on the term development of the SCNT rabbit embryos, and the possibility of the pregnancy maintenance of clones by cotransfer of PA embryos. The SCNT embryos were produced by fusing cumulus cells with enucleated cytoplasts before activation by electrical stimulation, and Dimethylaminopurine (6-DMAP) and Cyclohexamide (CHX) treatments. They were cultured in EBSS-complete medium regardless of their treatment with or without TSA. In vitro developmental data showed no differences in the cleavage and the blastocyst rates, and the blastocyst cell number between the TSA-treated and the untreated SCNT embryos. Two of the six recipients became pregnant after the embryo transfer (ET) in the TSA-treated group, and one pregnant female delivered seven live and three stillborn pups. The death of all live pups occurred within an hour to 19 days. Four of the seven recipients became pregnant in the TSA-untreated group. Three of them gave birth to six live and eight stillborn pups. Four pups of the TSA-untreated group have grown into adulthood, and three of them produced progeny. Cotransfer of three to four PA embryos with 26-32 SCNT embryos to the same recipient resulted in pregnancy and birth rates statistically no different compared to the control SCNT ET group. In conclusion, our results indicate that TSA treatment has a limited effect on the in vitro development of the SCNT embryos; furthermore, both the TSA-treated and the untreated clones can develop to term in rabbits, but none of the offspring from TSA-treated embryos survived to adulthood in our experiment.

Littermate Influence on Infant Growth in Mice: Comparison of SJL/J and ICR as Cotransferred Carrier Embryos

“…For recovering cloned mouse embryos derived from somatic cell nuclear transfer, cotransfer of 2-cell iCR embryos effectively promotes implantation and subsequent embryonic development [15,16]. Similar positive effects of cotransfer have been reported for cloned embryos from the pig, rat, and rabbit [13,14,20]. These effects might be attributable to the fact that, in multiparous animals, a minimum number of healthy embryos is required to trigger and maintain pregnancy.…”

Genetic Modification of Domestic Animals for Agriculture and Biomedical Applications

“…It has been demonstrated that histone deacetylase inhibitor treatment after SCNT can improve both in vitro development of SCNT embryos to the blastocyst stage and in vivo development to term following embryo transfer Cervera et al, 2009;Zhao et al, 2009aZhao et al, , 2010Das et al, 2010;Himaki et al, 2010a;Miyoshi et al, 2010). However, these treatments may be with some level of toxicity, as one group has reported that offspring from TSA-treated rabbit embryos did not survive to adulthood (Meng et al, 2009). The exact mechanism by which the HDACi treatment significantly improves the cloning efficiency remains largely unknown, although it has been shown to increase levels of global histone acetylation after HDACi treatment which may subsequently change the structure of chromatin and improve nuclear reprogramming (Shi et al, 2008;Iager et al, 2008;Das et al, 2010).…”

Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons

“…Contrary to mouse, swine and ruminants, the efficiency of somatic cell nuclear transfer (SCNT) in rabbits using standard methods is very low, regardless of the type of nuclear donor cells, and only a negligible percentage of offspring remain healthy and reach sexual maturity (puberty) [14][15][16][17] . Therefore, this method has limited practical use in rabbit transgenesis.…”