恒河猴tPA基因的克隆、测序与真核表达

本试验采用高浓度乙二醇（EG-8）作为胚胎冷冻保护剂，以高浓度的半乳糖作为解冻稀释液，对MMTV-Wnt-1转基因小鼠的早期囊胚进行玻璃化冷冻保存。结果在EG-2中平衡4-6min和解冻时间为3-5min时获得了最佳冷冻效果。此条件下冷冻胚胎的总回收率为88.61%（109/123）；回收胚胎在体外发育率达88.99%（97/109）；孵化率达81.65%（89/109）；发育胚胎的移植出生率为38.36%（69/181）；冷漠胚胎移植出生后经PCR检测一半小鼠整合阳性。结果表明使用高浓度的乙二醇作为冷冻保护剂和以高浓度的半乳糖作为冻稀释液能有效地保存转基因小鼠胚胎。

Cloning of Rhesus Monkey tPA cDNA and Its Expression in CHO

The high concentration ethylene glycol (EG-8, 8 mol/L) and galactose(1.7 mol/L) were used as a cryoprotectant and thawing diluents for embryo cryopreservation by vitrification in MMTV-Wnt-1 transgenic mice. The best results of cryopreservation were obtained if those embryos kept balance in EG-2 (2mol/L) for 4~6 minute before freeze and thawing 3~5 minute in galactose. The percentage of embryos cryopreserved and retrieve is 88.61%(109/123); there is 88.99%(97/109) of these embryos develop and 81.65%(89/109) developing to hatch blastocysts. After these development embryos were transfer to pseudocyesis mouse, 38.36%(69/181) developed to live young, and a half of these is positive mouse on the PCR test. It follows that high concentration ethylene glycol and galactose as a cryoprotectant for cryopreseveration by vitrification is effective.