精子与慢病毒共孵育条件的优化以及转基因猪的制备

【目的】探索慢病毒与精子共孵育的方法，并制备转基因猪。【方法】采用正交试验分析精子密度、病毒量、精子与慢病毒共孵育时间、精子与慢病毒共孵育温度对精子活力的影响，采用PCR和Southern blotting检测转基因。【结果】①优化慢病毒与精子共孵育条件为：100 µL慢病毒原液（5×105cfu）与1.0×107个/mL精子在17℃下孵育30 min；②与病毒孵育后的精子（109个/mL）给母猪人工授精时，经PCR检测49头仔猪中有14头呈阳性；③Southern blotting结果表明，8头PCR阳性仔猪中，4头融合了外源基因。【结论】优化了精子与慢病毒共孵育的条件（100 µL慢病毒原液（5×105cfu）与1.0×107个/mL精子在17℃下孵育30 min），成功制备了转基因猪，并检测有外源基因的融合。

Optimization of Incubation of Sperm and Lentivirus and Transgenic Pigs Production

【Objective】A new method was developed for producing transgenic pig using incubation of lentivirus and sperm. 【Method】Effects of sperm number, amount of lentivirus, incubation time and incubation temperature on sperm activity were assessed by the orthogonality design. PCR and southern blotting were used to analyze the transgene.【Result】The optimized incubation was incubation of 100 µL lentivirus (5×105cfu) with 1.0×107 sperms per mL for 30 min at 17℃. Using this procedure sperms were artificially inseminated into sows. PCR was used to detect the offspring. Fourteen of 49 (14/49) piglets were PCR-positive. Four of 8 PCR-positive pitlets showed foreign gene intergration revealed by using southern blotting.【Conclusion】Incubation indexes of lentiviruse and sperm optimized in this study are that 100 µL lentivirus (5×105cfu) with 1.0×107 sperms per mL for 30 min at 17℃, and transgenic pigs were produced by using this procedure. Foreign gene intergration were revealed.