Hirabayashi M, Takahashi R, Sekiguchi J, Ueda M
YS New Technology Inst. Inc. Tochigi, Japan.
Exp Anim. 1997 Apr;46(2):111-5. doi: 10.1538/expanim.46.111.
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.
研究了转基因大鼠冻融胚胎的体内活力，以及转基因在新生大鼠中的传递和表达。使用了三株转基因大鼠，它们携带与人生长激素基因相连的牛α-乳白蛋白基因（αLA/hGH）、牛β-酪蛋白基因（βCN/hGH）或牛α-S1酪蛋白基因（αS1CN/hGH）的启动子区域下游的基因。将二细胞期胚胎（非转基因Wistar雌性×杂合转基因雄性）置于10%（v/v）二甲基亚砜（DMSO）溶液中，以-0.5℃/min的速度从-7℃冷却至-30℃，然后投入液氮中。储存2至4年后，胚胎通过快速复温解冻。完整的胚胎被移植到第1天假孕受体的输卵管中。所有三株转基因品系的冻融胚胎解冻后存活率都很高（88%至92%），这与对照（非转基因）冻融胚胎的存活率（95%）相似。来自这三株品系的胚胎移植后发育成新生大鼠的比例（64%至68%）也与对照胚胎（60%）相似。在新生大鼠尾巴组织的DNA提取物中检测到了这些转基因（αLA/hGH、βCN/hGH和αS1CN/hGH），但其传递率（分别为41%、23%和32%）低于孟德尔遗传方式预期的50%。在携带αS1CN/hGH的转基因品系中，冻融胚胎及其转基因后代的转基因新生大鼠乳汁中hGH的分泌水平与它们的奠基鼠相同，为mg/ml水平。因此，转基因大鼠胚胎的两步冷冻是长期冷冻保存转基因的有效方法。
