卵巢打点注射质粒pIRES2-EGFP生产转基因兔的研究

本研究以绿色荧光蛋白为报告基因,转染兔卵母细胞,探索建立操作方便、效率高、成本低、可定量生产转基因兔的可行性方法.选择14只成年新西兰雌兔,每侧卵巢内分别打点注射0.25 mL 0.5～0.8 mg/mL的质粒,注射后第3日、第16日和第31日进行人工辅助配种;卵巢注射后第3日、第16日和31日随机各取1只雌兔卵巢做冰冻切片,置荧光显微镜下观察绿色荧光;应用PCR 和 Southern 杂交检测转基因新生仔兔阳性率.结果经荧光显微镜观察,卵巢注射后第3日配种的雌兔卵巢冰冻切片及48 h以后的胚胎呈现绿色荧光;卵巢注射报告基因后第3日配种的雌兔后代阳性率最高,PCR 和 Southern 杂交检测结果分别为72.7 %和45.5 %;第31日配种的后代阳性率最低,分别为36.8 %和15.8 %;第16日配种、第3日配种和第31日配种雌兔的阳性后代数量呈递减趋势.卵巢注射后第3日配种,虽然后代阳性率较高,但是其产仔数最少.实验结果表明,用注射器直接对卵巢打点注射外源基因生产转基因兔的方法操作简便、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.

Efficient Method to Generate EGFP Transgenic Rabbits by Multi-point Ovary Injection

The purpose of this study was to establish an easily operated,high-efficiency,low-cost method of generating transgenic rabbits.Enhanced green fluorescence protein (EGFP) was used as the reporter gene in order to test the efficiency of our protocol.We choosed fourteen healthy multiparous female New Zealand rabbits (8 to 12 months of age),weighing 3.0 to 3.2 kg,were selected and divided into three groups.Each rabbit received multi-point injections to both sides of the ovaries with EGFP plasmid at concentrations of 0.25 mg (0.5-0.8 mg/mL).Three days after injections,Group 1 females were mated with males,and 16 days after injections,Group 2 females were mated with males,and 31 days after injections,Group 3 females were mated with males.Two days after setting up the mating,one female rabbit from each group was randomly selected and green fluorescence was observed in their ovary tissues.After this,newborn transgenic rabbits were genotyped by PCR and Southern blotting.In Group 1,green fluorescence was observed in the frozen ovary section and embryo 48 hours after mating.Further,the offspring from this group had the highest positive rate for EGFP transgene expression by PCR (78.26 %) and Southern blotting (69.57 %).The offspring from Group 3 had the lowest positive rate by PCR and Southern blotting (41.18 % and 11.76 %,respectively).The number of positive offspring shows a decreasing trend for female rabbits mated on days 16,3,and 31,respectively.Although the offspring from female rabbits had the highest positive rate in Group 1,there were fewer newborn;there were also fewer positive individuals than the offspring from female rabbits mated on day 16.Therefore,we found post-operative day 16 to be the best for mating.The results showed that direct injection of exogenous gene into ovaries is a simple,efficient method to produce transgenic rabbits and provide production transgenic animals with a reference result.