脂质体介导外源基因体外转染山羊精子的研究

实验以山羊精子为对象,研究脂质体介导精子转染外源基因的效率和对生产阳性胚胎的影响.山羊鲜精离心洗涤除去精浆,分别用地高锌(DIG)末端标记的线形外源DNA与精子直接共育(对照组),阳离子脂质体LipofectamineTM-DNA复合体与精子共育(实验组),用免疫组化的方法分别检测它们的转染效率;用成熟卵母细胞与转染外源DNA并已获能的精子行体外受精,PCR方法检测生产阳性胚胎的效率.结果发现添加阳离子脂质体对提高山羊精子结合外源DNA和生产阳性胚胎的效率不显著(p＞0.05),对照组和实验组阳性精子率、DNase I消化后内化率与胚胎PCR阳性率分别为(47.56±3.09)%和(42.54±6.08)%、(26.67±2.40)%和(24.33±2.67)%、24.24%和22.73%,同时还发现添加阳离子脂质体对山羊精子活力和体外受精能力影响不显著(p＞0.05).

Abstract：

The potential use of liposome in sperm cells as vectors to transfer exogenous DNA via the fertilization of oocytes into the germ line of goat (Capra hircus) was evaluated. Seminal ejaculates collected artificially from healthy adult bucks were pooled together and the washed sperm were incubated with exogenous DIG-labeled linear DNA for 60 min (control group, CG), or with LipofectamineTM-DNA complex (treatment group, TG). The transfection rate of sperm was observed by the in situ hybridization methods. The in virto produced embryos were screened by PCR assay. The results showed that the improvement efficiency of sperm transfection with exogenous DNA by the addition of cationic liposome was nonsignificant, statistically, the positive sperm rate, internalization rate after DNase I treatment and PCR-positive rate of the embryo of CG and TG being (47.56±3.09) % and (42.54±6.08) %, (26.67±2.40) %and (24.33±2.67)% and 24.24% and 22.73%, respectively. The sperm motility and the fertilization rate were not significantly different between CG and TG (P＞0.05), suggesting that DNA transfer into goat sperm was not enhanced very efficiently in the presence of liposome.

Liposome-Mediated Exogenous DNA Uptake by Goat Sperm Cells