受体胚龄对鸡原始生殖细胞移植后归巢的影响

【目的】探究受体胚龄对鸡原始生殖细胞(Primordial germ cell,PGC)移植后归巢的影响,确定最佳回注时间窗口,为提高转基因鸡的生产效率提供参考依据。【方法】从广西黄羽鸡胚分离获得PGC,体外培养并进行转基因操作后,移植回注到不同胚龄的受体鸡胚中,移植5 d后分离性腺拍照观察,对比受体鸡胚孵育至14HH~17HH时对PGC迁移归巢到性腺的影响。【结果】分别在14HH~17HH胚龄时移植回注PGC,移植5 d后的鸡胚死亡率为30.0%~60.9%,但不同移植胚龄间的差异不显著(P>0.05)。根据鸡胚性腺中的GFP阳性细胞数可划分为6个等级(I~VI),且随着移植胚龄的增加,PGC迁移效率呈下降趋势。14HH胚龄移植的鸡胚性腺GFP阳性细胞集中在V级(40.0%),15HH胚龄移植的集中在III级(42.1%),16HH和17HH胚龄移植的则集中在II级(45.4%和54.5%),GFP阳性细胞含量最高等级(VI级)仅出现在14HH胚龄移植的鸡胚性腺中。【结论】随着受体胚龄的增加,PGC迁移效率呈下降趋势,但鉴于实际操作的难易程度,确定PGC移植回注的最佳时间窗口为14HH鸡胚(孵育50 h)。

Effect of recipient embryos age on homing of chicken primordial germ cell after transplantation

Objective]The present experiment was conducted to investigate effect of recipient embryo age on homing of chicken primordial germ cell ( PGC ) after transplantation and determine optimal reinjection time window , in order to provide basis for productivity of transgenic chicken. [Method]PGCs were isolated from embryos of native yellow feather chicken in Guangxi, and cultured in vitro, then genetically modified, finally transferred back into recipient embryos. On the 5th days after transplantation, the gonads of recipient embryos were isolated and imaged to compare effect of recipient embryo age(14HH-17HH) on PGC migration to gonads. [Result]The mortality rate of embryo ranged from 30.0% to 60.9% after transplanting PGC at embryonic age of 14HH-17HH, with non-significant differences(P>0.05). According to number of migrated GFP-positive cells , the gonads were classified into six grades ( I-VI ) . With the increase of embryo age , PGC migration efficiency decreased gradually. When transplanting PGC at age of 14HH , the GFP-positive cells in gonads were mostly grouped in Grade V ( 40 . 0%); when transplanting PGC at the age of 15HH , the GFP-positive cells in gonads were mostly grouped in Grade III (42.1%); when transplanting PGC at the ages of 16HH and 17HH, the GFP-positive cells in gonads were mostly grouped in Grade II(45.4% and 54.5%). The highest grade of gonads(VI) appeared only in the transplantation of PGCs at the age of 14HH. [Conclusion]With the increase of embryo age, PGC migration efficiency decreased gradually. In view of difficulty level in actual operation, the optimum time window for chicken embryo used in PGC transplantation is at the age of 14HH(incubation for 50 h).