小鼠半胚冷冻的研究

Freezing of Mouse Demi-emdryos

对半胚冷冻的方法和条件进行了系统研究,证明采用下述程序冷冻小鼠半胚可显著提高解冻半胚的成活率:①在含10％甘油的磷酸缓冲液中平衡35～40min;②在-6.5℃或-7℃诱发结晶,③以0.05℃/min的速度缓慢降温15min;④以0.3℃/min的速度降温至-35℃投入液氮;⑤在30～35℃的水浴中解冻;⑥在含不同浓度(6.6％,3.3％,0％)甘油和0.3mol/L蔗糖的磷酸缓冲液中平衡35～40min,去除甘油。将按上述程序冷冻—解冻的16对半胚移植后,获半胚鼠12只,其中5对为同卵双生。

The methods and conditions suitable to freezing of demi-embryos were systematically studied. The results showed that freezing of demi-embryos embedded with agar can improve the survival rate of frozen-thawed mouse demi-embryos using the following procedure; (l)equilibration in PBS with 10% glycerol for 35-40min; (2 )seeding al-6.5℃ or-7℃; (3 )slow cooling at about 0.05/min for 15 min; ( 4 )continuous cooling at 0.3/min to-35 at which demi-embryos with agar were plunged into liquid nitrogen; (5)thaw-ing in water at 30-35℃; (6)removal of glycerol by equilibration in PBS With 6.6%, 3.3%, 0% glycerol and 0.3 mo 1/L sucrose for 40(10, 15, 15) min. Sixteen pairs of derai-embryos frozen-thawed by the above procedure were transferred, of 8 recipients, 5 became pregnant(62.5%)and produced 12 young (37.5%) at delivery, including 5 pairs of monozygotic twins(31.3%).