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目前建立转基因动物最常用的方法仍然是经典的受精卵显微注射法.但这一方法存在诸多的不足之外:所需的仪器设备价格昂贵;技术要求高;成功率低.电穿孔导入法,是通过高压电场使细胞膜通透性发生暂时性的可逆变化,从而使DNA分子进入细胞内的一种物理方法.目前已有一些穿孔导入法促使外源基因转染精子的报道,但由于各处实验室所使用的电击仪器不同,电击缓冲液及电击条件也不相同,因此,各实验结果的报道并不一致.我们用假阴道法采集家兔精子,经电击缓冲液洗涤3次,调整精子浓度至10~6个/ml.电击悬液总体积100ml,加入环状重组质粒pV-WAP-tPA(浓度20μg/ml).使用BAERON600型基因转移仪,设电击条件:周期10;脉冲数1000;脉冲宽度100;距离2.0mm;电压2000,4000,6000;脉冲时间500,1000,1500ms.另设电击悬液中不加质粒作
At present, the most commonly used method of establishing transgenic animals is still the classical microinjection method of fertilized eggs, but there are many deficiencies in this method: the required equipment is expensive, the technical requirements are high, the success rate is low, and the electroporation method , Is a physical method of transiently reversing the permeability of a cell membrane through a high-voltage electric field so that the DNA molecules enter the cell.At present, some perforation methods have been reported to facilitate the transfection of foreign genes into sperm, At the laboratory using different electric shock instruments, shock shock and shock conditions are not the same, therefore, the experimental results reported are not consistent.We collected fake vagina rabbit sperm, washed three times by electric shock buffer to adjust the sperm The concentration to 10 ~ 6 / ml. The total volume of the shock suspension 100ml, adding a circular recombinant plasmid pV-WAP-tPA (concentration 20μg / ml) using BAERON600 gene transfer device, set the shock conditions: cycle 10; ; Pulse width 100; distance 2.0mm; voltage 2000,4000,6000; pulse time 500,1000,1500 ms.