In vitro formation of tetraploid rat blastocysts after fusion of two‐cell embryos

Abstract

Gene targeting technology is not available in the rat which is an animal model of major importance, e.g., in cardiovascular research. This is due to the fact that the rat embryonic stem cell (ESC)-like cells established by several groups do not form germ-line chimeras when injected into blastocysts. In the mouse, the aggregation of ESC with tetraploid embryos has allowed the generation of animals completely derived from these cells. However, aggregation of rat ESC-like cells with tetraploid rat embryos has not yet been attempted to evaluate their developmental capacity. Therefore, we established a method to produce tetraploid rat embryos by fusion at the two-cell stage. Chemical fusion by polyethylene glycol (PEG) was shown to be less efficient (56.3% fused embryos) than electrofusion (96.1% fused embryos). The rate of development of fused embryos to blastocysts was independent of the fusion method and similar to the rate of control embryos. However, this rate was lower when the embryos had been cultured from the zygote state before fusion (14-20%) compared to freshly isolated two-cell embryos (41-63%). Alike for the mouse, blastocysts derived from fused two-cell rat embryos contained about half the number of cells as control blastocysts and were homogeneously tetraploid with no evidence of mosaicism. This method may be useful for the establishment of gene-targeting technology in the rat.

Tetraploid development in the mouse

“…Based on comparisons with controls, and the observation that diploid cells tend to display a strong competitive advantage in 4n:2n chimeras (see below), the conclusions are likely to be valid. While electrofused blastomeres of the rat and pig are were reported to develop as homogeneously tetraploid embryos (Prather et al, 1996;Krivokharchenko et al, 2002), rabbit and bovine embryo electrofused at the two-cell stage displayed occasional (rabbit) and frequent (cow) mosaic (4n:2n) preimplantation development for unknown reasons (Ozil and Modlinski, 1986;Iwasaki et al, 1989;Curnow et al, 2000). In light of this, it may be prudent to consider the possibility that other strains of mice may not behave as those detailed above (Table 1).…”

Tetraploidy Induced by Electrofusion - A Review

“…Krivokharchenko et al (2002) observed 96% fusion rate after electrofusion of 2-cell stage embryos in rat when used 0.6 kV/cm DC pulses for 20 μs duration.…”

“…Fusion of embryos induced by electrical pulses was used for production of tetraploid embryos in different species including mouse (Sekirina et al, 1997), rabbit (Ozil and Modlinski, 1986), pig (He et al, 2013), bovine (Darabi et al, 2008) and rat (Krivokharchenko et al, 2002).…”

“…At the blastocyst stage of the electrofused embryos of rats and mice, the uniform tetraploidy was documented (Krivokharchenko et al, 2002).…”

Efficient Production of Bovine Tetraploid Embryos by Electrofusion of In Vitro Produced Two-Cell Stage Embryos

“…It is also possible to produce parthenogenetic tetraploid embryos by inhibiting the formation of both first (1 st PB) and second polar bodies (2 nd PB) with cytochalasin B or other cytokinesis-inhibition drugs during parthenogenetic activation of oocytes [16,17]. The third method is to fuse two diploid blastomeres at the 2-cell stage embryo with either polyethylene glycol (PEG) [18,19], inactivated Sendai virus [20] or by electrofusion. Among these fusion methods, electrofusion is the most widely used tool for the production of tetraploid embryos [1,21,22] as it is safer than the chemical-or virus-mediated methods and more convenient and economical than microsurgical injection [14].…”