改良背主动脉注射法对鸡胚发育与转基因表达效率的影响

Effects of Modified Dorsal Aorta Injection on Development and Transgene Expression in Chick Embryos

背主动脉注射是生产转基因鸡的经典方法,该方法不需换壳培养,但壳内注射技术难度大,并且无法实时观察后期胚胎发育情况。本实验对背主动脉注射法进行了改进,将增强型绿色荧光蛋白(EGFP)腺相关病毒(Adeno-associated Virus,AAV)壳外注射至150枚HH 14~16期(Hamburger-Hamilton Stage 14~16)鸡胚背主动脉中,再进行换壳培养,继续孵化至出壳,观察和分析改良背主动脉注射法与传统背主动脉注射法和胚盘下腔注射法对发育8、14、18、21 d鸡胚存活率、孵化率以及EGFP阳性检出率的影响。结果表明:改良背主动脉注射组鸡胚存活率均高于传统背主动脉注射组与胚盘下腔注射组(P<0.05或P<0.01);改良背主动脉注射组的鸡胚孵化率(37%)高于传统背主动脉注射组(16%)与胚盘下腔注射组(28%)(P<0.01);荧光蛋白手电筒检测显示,改良背主动脉注射组鸡胚EGFP阳性率(17%)明显高于传统背主动脉注射组(13%)与胚盘下腔注射组(12%)(P<0.01)。综上,背主动脉壳外注射结合换壳培养提高了转基因鸡胚胎孵化率和EGFP阳性检出率,对提高转基因鸡效率具有重要价值。

Dorsal aorta injection is a classical method for producing transgenic chickens.The advantage of this method is that there is no need to surrogate eggshell culture,but the disadvantage is that it is difficult to perform injection inside eggshell,and the late development of embryos cannot be observed in real time.In this experiment,the dorsal aorta injection method was modified.Adeno-associated virus(AAV)was injected to dorsal aorta outside eggshell,followed by surrogate eggshell culture until to hatch.The AAV injection was simultaneously conducted with classic dorsal aorta injection and subgerminal cavity injection,and effects of the three injection methods were compared on the rates of survival or hatch and EGFP expression in chicken embryos at age of 8,14,18 and 21 days.The results showed that the survival rate of chicken embryos in the modified dorsal aorta injection group was higher than that in the classic dorsal aorta injection group and the subgerminal cavity injection group(P<0.05 or P<0.01);the hatch rate of chicken embryos in the modified dorsal aorta injection group(37%)was higher than that in the classic dorsal aorta injection group(16%)and the subgerminal cavity injection group(28%)(P<0.01);the fluorescent protein flashlight detection showed that the EGFP+rate in chicken embryos in the modified dorsal aorta injection group(17%)was significantly higher than that in the classical dorsal aorta injection group(13%)and the subgerminal cavity injection group(12%)(P<0.01).All results illustrated that dorsal aorta injection combined with surrogate eggshell culture is an innovative method,which improves the hatchability and EGFP expression efficiency of putative transgenic chickens,and will contribute to promoting the efficiency of transgenic chickens.