睾丸和卵巢打点注射pIRES2-EGFP质粒生产转基因兔的研究(英文)

为探讨直接用注射器对睾丸和卵巢打点注射pIRES2-EGFP质粒转染兔的精子细胞和卵母细胞,建立操作方便、效率高、成本低、可批量生产转基因兔的可行性。选择5只成年新西兰雄兔、29只经产新西兰雌兔,每侧睾丸和卵巢内分别打点注射0.5~0.8 mg/mL质粒0.25 mL。①第3周随机取一只雄兔睾丸做冰冻切片,于荧光显微镜下观察转染情况。②(Ⅰ:1#♂×7♀、Ⅱ:2#♂×7♀、Ⅲ:3#♂×7♀、Ⅳ:4#♂×7♀)其它雄兔于第6周和第7周与3日前做过卵巢注射并超排处理的雌兔配种,最后采集新生仔兔耳组织,提取基因组DNA,经PCR和Southern杂交检测阳性率。结果显示雄兔睾丸冰冻切片于荧光显微镜下可见绿色荧光,统计所得后代转基因阳性率,并进行差异分析,其中Ⅰ组后代阳性率最高,PCR和Southern杂交检测阳性率为93.55%和83.87%,Ⅳ后代阳性率最低,依次为80.65%和70.97%,但是各组之间差异不显著。上述实验结果表明,对睾丸和卵巢同时注射外源基因可获得较高阳性率的转基因后代,生产转基因的方法操作简便、高效,为日后大规模制备一些大型家畜的转基因后代奠定了基础。

Research on Production of Transgenic Rabbits by Multi-point Injection of Plasmid pIRES2-EGFP to Testis and Ovary

To explore and establish easy, highly efficient, low-cost methods for mass production of transgenic rabbits through transfection of the rabbit spermoblast and oocyte by multi-point injection of plasmid pIRES2-EGFP into testis and ovary. We choosed 5 mature male New Zealand rabbits and 29 multiparous female New Zealand rabbits were selected and subjected to multi-point injection of plasmid (0.25 mL of 0.5-0.8 mg/mL plasmid solution) into the testis and ovary on both sides. Test 1: In week 3, frozen sections of testis were made from one randomly selected male, and the transfection was observed under a fluorescence microscope. Test 2: ( GroupⅠ: 1#♂×7♀rabbits; GroupⅡ: 2#♂×7♀rabbits; GroupⅢ: 3#♂×7♀rabbits; GroupⅣ: 4#♂×7♀rabbits. ) Female rabbits underwent plasmid injections and were superovulated three days prior to mating with male rabbits. Ear tissue from the resulting newborn rabbits was collected, genomic DNA was extracted, and transgenic individuals were identified by PCR and Southern blotting. The results showed that, Green fluorescence could be observed under a fluorescence microscope for the frozen sections of testis. After counting transgenic offspring produced and performing variance analysis, Group I showed the highest rate of transfection (93.55 % and 83.87 % based on PCR and Southern blot analyses, respectively). Group IV showed the lowest rate of transfection (80.65 % and 70.97 %, respectively); however, the differences between groups were not significant. 　These experimental results showed that greater numbers of transgenic offspring could be obtained through injection of an exogenous gene into testis and ovary at the same time. The method was simple, efficient, and could enable large-scale preparation of transgenic offspring of large domestic animals in the future.