Significant Improvement in Cloning Efficiency of an Inbred Miniature Pig by Histone Deacetylase Inhibitor Treatment after Somatic Cell Nuclear Transfer1

Abstract

The NIH miniature pig was developed specifically for xenotransplantation and has been extensively used as a large animal model in many other biomedical experiments. However the cloning efficiency of this pig is very low (less than 0.2%) and this has been an obstacle to the promising application of these inbred swine genetics for biomedical research. It has been demonstrated that increased histone acetylation in somatic cell nuclear transfer (SCNT) embryos, by applying histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA), significantly enhances the developmental competence in several species. However some researchers also reported that TSA treatment had various detrimental effects on the in vitro and in vivo development of the SCNT embryos. Here we report that treatment with 500 nM Scriptaid, a novel HDACi, significantly enhanced the development SCNT embryos to the blastocyst stage when NIH inbred fetal fibroblast cells (FFCs) (21% vs. 9%, P < 0.05) were used as donors compared to the untreated group. Scriptaid treatment resulted in 8 pregnancies from 10 embryo transfers (ET) and 14 healthy NIH miniature pigs from 8 litters while no viable piglets (only 3 mummies) were obtained from 9 ETs in the untreated group. Thus Scriptaid dramatically increased the cloning efficiency when using inbred genetics from zero to 1.3%. In contrast, Scriptaid treatment decreased the blastocyst rate in IVF embryos (from 37% to 26%, P < 0.05). In conclusion, the extreme low cloning efficiency in the NIH miniature pig may be caused by its inbred genetic background and can be improved by alteration of genomic histone acetylation patterns.

Bone Marrow Mesenchymal Stem Cells Are an Attractive Donor Cell Type for Production of Cloned Pigs As Well As Genetically Modified Cloned Pigs by Somatic Cell Nuclear Transfer

“…the efficiency of 1.3-2.4%, even though they used Fs as a donor cells Zhao et al, 2009). Because cloned embryos were treated with drugs that can improve reprogramming prior to embryo transfer in the Zhao et al study, and preselected high-quality oocytes were used for cloning in the Kim et al study, these could be the main reasons causing the difference in cloning efficiency between our study and their studies.…”

Piglets cloned from induced pluripotent stem cells

“…We thus thought that this may underlie the discrepancy of in vivo developmental capacity of NT embryos derived from mouse iPSCs and piPSCs. Previous studies have also demonstrated that the state of histone acetylation in somatic cell NT embryos, significantly impacts the developmental competence in several species [9].…”

“…Therefore, in the second round of experiments, we took the following measures to attempt to improve the developmental capacities of cloned embryos: 1) silence the exogenous transcription factors through spontaneous differentiation of piPSCs before they are used as donor cells; 2) increase histone acetylation by treating the constructed embryos with Scriptaid, a novel histone deacetylase inhibitor that can improve cloning efficiency through increasing transcriptional activity [9]. To silence exogenous transcription genes, piPSCs were allowed to spontaneously differentiate for 4-6 days.…”

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

“…FFs were established as previously described (Kragh et al, 2005;Onishi et al, 2000;Zhao et al, 2009). Briefly, fetuses obtained 40 days after insemination were decapitated, eviscerated, and cut into small pieces with fine scissors in phosphate-buffered saline (PBS).…”