慢病毒法生产转基因鸡的初步研究

Production of Transgenic Chickens with Lentivirus Method

[目的]探索利用慢病毒生产转基因鸡的适宜方法。[方法]以增强型绿色荧光蛋白为报告基因构建慢病毒载体,包装病毒后感染鸡早期受精卵,并检测不同注射操作和不同接种滴度对鸡胚存活率的影响,同时对接种病毒鸡胚的GFP表达情况进行检测。[结果]采用105、106和107IU/ml的慢病毒滴度,鸡胚孵化率分别为87%、72%和66.7%。以雏鸡血液基因组为模板PCR鉴定转基因阳性率为45.5%;冰冻切片技术发现GFP基因在鸡体内不同组织的表达强度差异显著,免疫系统内表达最强,生殖系统和肌肉组织中GFP基因无表达。[结论]试验初步筛选出较适宜的慢病毒感染滴度,该结果为研制蛋鸡生物反应器和转基因鸡抗病新品系奠定了基础。

[Objective] The aim was to explore optimal method for transgenic chickens production with lentivirus.[Method] Lentiviral vector was constructed with enhanced green fluorescent protein（EGFP） and fertilized ova at early stages were inoculated after virus packaging.In addition,effects of injection and virus titer on survival rate of embryos were detected and GFP expressions of inoculated embryos were observed.[Result] The hatching rates of embryo were 87%,72% and 66.7% with titers of lentivirus at 105,106 and 107 IU/ml,respectively.Transgenic positive rate was 45.5% through PCR with genome of chick blood as template.Expression of GFP genes in different tissues differed significantly through frozen section technology,for example,expression in immune system was the highest and expression was not proved in genital system and muscular tissue.[Conclusion] The research primarily selected optimal lentivirus titer,laying basis for development of bioreactor（eggchicken） and the new transgenic disease-resistant chicken.