Viability of transgenic rat embryos after freezing and thawing

In-vivo viability of frozen-thawed embryos derived from transgenic rats, as well as the transmission and the expression of transgenes in the resultant newborn rats, was investigated. Three strains of transgenic rats, carrying human growth hormone gene connected downstream to the promoter region of the bovine alpha-lactalbumin gene (alpha LA/hGH), bovine beta-casein gene (beta CN/hGH) or bovine alpha-S1 casein gene (alpha S1CN/hGH), were used. Two-cell stage embryos (non-transgenic Wistar female x heterozygous transgenic male) were placed in 10% (v/v) dimethylsulfoxide (DMSO) solution and cooled from -7 to -30 degrees C at -0.5 degree C/min before being plunged into liquid nitrogen. After 2 to 4 years storage, the embryos were thawed by rapid warming. The intact embryos were transferred into the oviducts of Day 1 pseudopregnant recipients. The postthaw survival rate of frozen embryos was high in all 3 transgenic strains (88 to 92%), which was similar to that of control (non-transgenic) frozen embryos (95%). Development to newborn rats following transfer of embryos derived from the 3 strains (64 to 68%) was also similar to that of control embryos (60%). These transgenes (alpha LA/hGH, beta CN/hGH and alpha S1CN/hGH) were detected in the DNA extracts from tail tissue of the newborn rats, but the transmission rates (41, 23 and 32%, respectively) were lower than 50% which is expected in the Mendelian fashion. In a transgenic line carrying alpha S1CN/hGH, hGH levels of secretion into the milk of transgenic newborn rats derived from frozen-thawed embryos and her transgenic offspring were the same mg/ml-level as that of their founder rat. Two-step freezing of embryos derived from transgenic rats was therefore an effective method for the long-term cryopreservation of transgene.