冷冻对山羊精子转染外源DNA和体外制备转基因胚胎的影响

Effect of cryopreservation on the efficiencies of goat sperm in picking up exogenous DNA and resuted transgeneic embryos

本实验将鲜精和冻精分别与地高锌标记的线形化的pEGFP-N1质粒孵育转染,用原位杂交方法检测转染效率;PCR和Southern Blotting检测精子与外源DNA的整合效率;与成熟卵母细胞体外受精,PCR检测阳性胚胎比率,用透射电镜技术、碘化丙锭和羟化荧光素双探针技术和单细胞电泳(Single Cell Gel Electrophoresis,SCGE)技术,观察精子冷冻前后的超微结构、精子质膜完整性和精子核DNA损伤的变化,研究冷冻对山羊(Caprahircus)精子转染内化外源DNA和体外制备转基因胚胎的影响及机理。结果表明,冻精显著提高了转染外源DNA的效率(81.60%±16.59%vs32.95%±2.93%,t=4.873,P=0.003;41.80%±6.26%vs27.89%±8.64%,t=2.634,P=0.039)。PCR和Southern Blotting检测表明外源DNA已经整合到精子基因组上。用冻精与成熟卵母细胞体外受精,体外受精穿透率和卵裂率显著低于鲜精组(24.19%±3.15%vs58.86%±3.73%,t=7.131,P<0.001;11.83%±2.37%vs29.71±3.47%,t=4.302,P<0.001),但体外生产的胚胎PCR阳性率比鲜精组显著提高(45.45%±10.87%vs24.44%±6.06%,t=1.750,P=0.013)。超微结构观察和双荧光探针检测都发现冷冻-解冻精子质膜完整性降低(8.34%±4.21%vs65.67%±6.46%,t=12.492,P<0.001),SCGE显示冷冻极显著增加了精子彗尾长度和彗星细胞比例(42.67μm±4.56μmvs21.14μm±2.36μm,t=5.644,P=0.005;60.00%±4.00%vs17.37%±2.57%;t=15.787,P<0.001)。冷冻-解冻可以提高山羊精子转染外源DNA的效率,冷冻破坏精子质膜完整性,解除质膜的阻碍作用,是提高外源DNA转染效率的一个主要原因。

The efficiency and the ability of fresh and frozen-thawed sperm in picking up exogenous DNA were investigated in this study. The fresh and frozen-thawed sperm was incubated with linearized end-labeled pEGFP-N1 plasmid DNA, and detected by in situ hybridization method. The in vitro producted embryos were screened by PCR assay. The ultrastructure of activated spermatozoa were observed by transmission electron microscope （TEM）, and the integrity of sperm plasma membrane was evaluated with a combination of fluorescent probes-carboxifluoreseein diacetate and propidium iodide （PI）. The sperm genomic DNA damage was determined by single cell gel electricity assay （SCGE） methods. The results showed that the frozen-thawed treated goat sperm cells were more efficient and more reliable than untreated sperm in picking up exogenous DNA and subsequently internalizing the DNA into sperm nuclei （81.60% ± 16.59% vs 32.95% ± 2.93%, t = 4.873, P = 0.003; 41.80% ± 6.26% vs 27.89% ± 8.64% , t = 2.634, P = 0.039） . The exogenous DNA retained its integrity in sperm as demonstrated with PCR and Southern Blotting assay methods. The rate of transgenesis embryos produced by exogenous DNA incubated sperm was enhanced significantly （45.45% ± 10.87% vs 24.44% ± 6.06%, t = 1.750, P = 0.013）. The semen cryopreservation redueedthe integrity of sperm plasma membrane （8.34% ± 4.21% vs 65.67% ± 6.46%, t = 12.492, P 〈 0.001） and enhaneed the sperm genomie DNA strand breakage significantly. These results suggest that semen eryopreservatlon could enhanee the sperm ability in picking up foreign DNA. The cryopreservation-induced changes in the sperm plasma membrane faeilitate the internalization exogenous DNA [Acta Zoologica Sinica 54 （6）： 1089- 1097, 2008].