Combined refinements to somatic cell nuclear transfer methods improve porcine embryo development

Abstract

The discovery of how to utilize CRISPR (clustered, regularly interspaced, short, palindromic repeats)-Cas (CRISPR-associated) systems for genome modification has accelerated development of the field of genome editing, especially in large animals such as pigs. The low efficiency of somatic cell nuclear transfer (SCNT) is now becoming a major obstacle in the production of genome-edited animals via cell-mediated approaches and improving efficacy of this technique is crucial. In this study, we propose a few simple modifications to a zona-free SCNT protocol that are effective to produce numerous high-quality blastocysts. To refine the SCNT protocol we modified the following steps/factors: 1) culture medium for SCNT embryos, 2) chemical treatment to prevent precocious activation of the manipulated/reconstructed oocytes and 3) donor cell serum starvation treatment. Although changes in each of these steps only resulted in small improvements, the combination of all modifications altogether significantly enhanced developmental competence of SCNT embryos. Our modified method yielded approximately three times greater blastocyst formation rates. Moreover, resulting blastocysts had roughly twice as many cells as compared to blastocysts produced by the conventional SCNT method. With these significant in vitro improvements, our refined SCNT method is potentially suited for use in the production of genome edited pigs.

Keywords: Donor cells; Embryonic development competence; Pigs; Somatic cell nuclear transfer.

Fig. 1.

Day 7 somatic cell nuclear transfer (SCNT) blastocysts derived from adult fibroblasts from a wild boar produced according to the optimized protocol in culture (A) or after staining with DAPI (B). Arrows indicate blastocysts.

Fig. 2.

Western blotting analysis of tri-methylation of lysine 9 on histone 3 (H3K9me3) in fibroblasts from wild boar (FWB), in fibroblasts from Western crossbred pig (FLWD), and in cumulus cells from Western crossbred pigs. Cells were serum starved in serum-free DMEM for 2 days (L1), in 0.5% FBS DMEM for 5 days (L2) or were not serum starved (L3).