In vitro formation of tetraploid rat blastocysts after fusion of two-cell embryos.
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.
Krivokharchenko
Alexander
A
Max-Delbrück-Center for Molecular Medicine (MDC), Berlin-Buch, Germany.
Galat
Vassili
V
Ganten
Detlev
D
Bader
Michael
M
D000818:Animals; D001755:Blastocyst / Q000166:cytology* / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods; D002459:Cell Fusion / Q000379:methods*; D005260:Female; D018390:Gene Targeting / Q000379:methods; D011123:Polyploidy; D051381:Rats; D017208:Rats, Wistar / Q000196:embryology* / Q000502:physiology