Production of cloned sei whale (Balaenoptera borealis) embryos by interspecies somatic cell nuclear transfer using enucleated pig oocytes

Abstract

In this study, we examined the feasibility of using subzonal cell injection with electrofusion for interspecies somatic cell nuclear transfer (iSCNT) to produce sei whale embryos and to improve their developmental capacity by investigating the effect of osmolarity and macromolecules in the culture medium on the in vitro developmental capacity. Hybrid embryos produced by the electrofusion of fetal whale fibroblasts with enucleated porcine oocytes were cultured in modified porcine zygote medium-3 to examine the effects of osmolarity and fetal serum on their in vitro developmental capacity. More than 66% of the whale somatic cells successfully fused with the porcine oocytes following electrofusion. A portion (60 approximately 81%) of the iSCNT whale embryos developed to the two- to four-cell stages, but no embryos were able to reach the blastocyst stage. This developmental arrest was not overcome by increasing the osmolarity of the medium to 360 mOsm or by the addition of fetal bovine or fetal whale serum. Our results demonstrate that sei whale-porcine hybrid embryos may be produced by SCNT using subzonal injection and electrofusion. The pig oocytes partly supported the remodeling and reprogramming of the sei whale somatic cell nuclei, but they were unable to support the development of iSCNT whale embryos to the blastocyst stage.

Fig. 1

Karyotyping of the sei whale fetal fibroblasts used as donor cells to produce whale-porcine interspecies nuclear transfer embryos. Karyotype analysis shows a set of normal diploid chromosomes (2n = 44).

Fig. 2

Photomicrographic images of cloned sei whale embryos (A and B) derived from interspecies somatic cell nuclear transfer using pig oocytes as recipient cytoplasts. The metaphase-like structures of nuclei that arrested at the two- (C) and four-cell (D) stages after 7-day culture in a porcine zygote medium-3 medium containing 0.3% (w/v) bovine serum albumin. Scale bars : 20 µm.