用注射器直接对睾丸打点注射EGFP生产转基因兔的试验研究

Generation of Transgenic Rabbits by Testis Injection with Syringe

为探讨直接用1 mL注射器对睾丸打点注射pIRES2-EGFP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4只成年新西兰雄兔,双侧睾丸内分别打点注射0.5～0.8 mg/mL浓度的质粒0.25 mL,第3周和第8周分别取4^#和3^#雄兔睾丸做冰冻切片,置荧光显微镜下观察是否发绿色荧光。其它雄兔于第3周开始参与配种,最后采新生仔兔耳组织提取其基因组进行PCR和Southern杂交检测阳性率。结果表明,雄兔睾丸冰冻切片于荧光显微镜下可见绿色荧光,PCR和Southern杂交检测表明在睾丸注射后的第6周和第7周进行配种所得后代阳性率最高。不同雄兔后代经PCR和Southern杂交检测平均阳性率存在差异（P〈0.05）,2^#雄兔后代平均阳性率达到56.46%,3^#雄兔的最低达36.96%的阳性率。表明用注射器直接对睾丸打点注射外源基因生产转基因的方法操作简便、高效,为大规模制备一些大型家畜的转基因后代奠定了基础。

The evaluation to the feasibility of establishing transgenic rabbit by intratesticular injection pIRES2-EGFP with syringe which is convenient,efficient,cost little price,and produce rabbits in quantity was carried out.4 adult New Zealand male rabbits,0.5mg/ml-0.8mg/ml concentration of the plasmid DNA,0.25ml doses were injected into the bilateral testis.The rabbit＇s testis were collected in random for discerning fluorescence difference by frozen section of spermary and fluorescence microscopic image after three weeks,and eight weeks later do the same.Others male rabbits did copulation with female rabbits three weeks later,the positive offsprings were detected by PCR and Southern blot.The result showed that fluorescent light transfection rate of spermatozoa was highest.the result showed that more positive rabbits was born after intratesticular injection in 6th and 7th week.Different male rabbits had different progeny＇s positive ratio,which were identify by Polymerase chain reaction（PCR） and Southern blot were mated.The highest 2^# father＇s babies＇ equilibrational positive ratio was 56.46%,and 3^#＇s was lightest was 36.96%.Conclusion The result indicate that the method of establishing transgenic rabbit by intratesticular injection with syringe was convenient,high efficiency,which could be suitable for creating a large number of transgenic animals.especially,for producing domestic animals.